Characterisation of class II and III fructose …/media/worktribe/output-191544/...Ali Abdullah Almohaish A thesis submitted in partial fulfilment of the requirements of Edinburgh

Characterisation of class II and III fructose

bisphosphatases in Clostridium

acetobutylicum ATCC 824

Ali Abdullah Almohaish

A thesis submitted in partial fulfilment of

the requirements of Edinburgh Napier

University, for the award of Doctor of

Philosophy

March 2012

i

Abstract

In low GC Gram-positive bacteria, the phenomenon of carbon catabolite repression is

dependent on a metabolite-activated bifunctional protein kinase/phosphorylase. In

Clostridium acetobutylicum ATCC 824, the hprK gene encodes the bifunctional protein

kinase/phosphorylase that is dependent on fructose 1,6-bisphosphate for activity.

However, a putative glpX class II gene that might encode for fructose 1,6-

bisphosphatase is located upstream of hprK and to date this is a unique gene

arrangement. Therefore, the product of the putative glpX gene might have a vital role in

regulating the activity of bifunctional protein kinase/phosphorylase by hydrolysing

fructose 1,6-bisphosphate which is needed for the kinase activity. In the present work,

experimental evidence is presented that the putative glpX gene encodes a fructose 1,6-

bisphosphatase which can hydrolyse fructose 1,6-bisphosphate to fructose 6-phosphate

and phosphate. FBPase activity was first demonstrated by cloning and transforming

the glpX gene into an E. coli fbp mutant which cannot grow on gluconeogenic substrates

such as glycerol. The transformed glpX gene was able to complement the fbp mutation

of E. coli for growth on glycerol. GlpX was overexpressed as a GST-fusion protein and

purified, and activity was demonstrated using fructose 1,6-bisphosphate as substrate.

Activity was assayed at pH 8.0, and in the presence of Mn2+

, but the enzyme was

inhibited completely by 1 mM phosphate. A putative fbp class III gene was also

overexpressed and the encoded protein was purified as a GST-fusion product in order to

compare Fbp with GlpX. To our knowledge, this is the first study that was able to

purify a Fbp class III enzyme. The Fbp protein showed almost the same behaviour

towards inhibitors compared to GlpX, but had a considerably higher specific activity

than GlpX under the conditions of the experiments. The glpX gene was shown by RT-

PCR to be transcribed together with hprK on the same mRNA during growth on

glucose, and this indicates that glpX is unlikely to be a gluconeogenic gene. The results

suggest that GlpX may play a novel and specific role in regulating the kinase activity

activity of bifunctional protein kinase/phosphorylase and carbon catabolite repression in

C. acetobutylicum.

http://ukpmc.ac.uk/abstract/MED/18613233/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=1488&lvl=0

ii

Acknowledgement

This research project would not have been possible without the support of many people.

It is a pleasure to express my gratitude to them all in my humble acknowledgment. First

and foremost, I would like to express my sincere gratitude to my academic supervisor

Prof. Martin Tangney for the continuous support of my PhD study and research, for his

patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in

all the time of research and writing of this thesis. I could not have imagined having a

better supervisor and mentor for my PhD research.

Also, I would like to record my gratitude to my second academic supervisor Dr. Wilf

Mitchell for his supervision, advice, and guidance from the very early stage of this PhD

research as well as giving me extraordinary experiences throughout the work in the lab.

Most of all, he gave me constant encouragement and support in various ways which

contributed to create an excellent scientist that can handle novel researches. He gave

me the chance to help, guide and supervise many undergraduate and postgraduate

students and to present my work in prestigious conferences. I am really indebted to him

more than he actually knows.

I would also acknowledge my Saudi Aramco advisers Christine Evans for helping me to

get my extension and Saad Nassar, for his advice and support throughout my study. I

am very grateful to Dr. Eve Bird for being the first person who taught me basic skills in

microbiology. My special thanks go to my parents for giving birth to me in the first

place, supporting and praying for me to succed in this life. Finally, words fail me to

express my gratitude to my wife for her patience, support and persistent confidence in

me, which has reduced the load of the PhD on my shoulders.

iii

List of abbreviations

ABE Acetone Butanol Ethanol

ADP Adenosine diphosphate

Amp Ampicillin

ATP Adenosine 5’-triphosphate

bp Base pairs

cac (referring to gene on chromosome) C. acetobutylicum

CcpA Catabolite control protein A

CCR Carbon catabolite repression

cre

DNA

Catabolite repression HPr-like protein

Deoxyribonucleic acid

EI Enzyme One (phosphotransferase system)

EII Enzyme Two (phosphotransferase system)

FBP

FBPase

fbp

GlpX

glpX

g

GC

HPr

HPr K/P

hprK

Fructose 1,6-bisphosphate (sugar).

Any class of fructose 1,6-bisphosphatase enzyme

Referring to gene that encodes fructose 1,6-bisphosphatase

Referring to class II fructose 1,6-bisphosphatase

Referring to gene that encodes class II fructose 1,6-bisphosphatase

Grams

Guanine Cytosine content

Heat stable histidine-phosphorylatable protein

Bifunctional HPr protein kinase/ phosphorylase

Referring to gene that encodes HPr K/P enzyme

Lac (referring to operon) Lactose

LB Luria-Bertoni medium

L Litres

min Minutes

nm Nanometres

OD Optical Density

ORF Open Reading Frame

PEP PhosphoEnolPyruvate

PTS

P-Ser-HPr

PhosphoTransferase System

HPr phosphorylated at a serine site

iv

P-His-HPr

S

U

µg

HPr phosphorylated at a histidine site

Second

units

Micrograms

V Volts

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

v

Table of Contents Abstract……………………………………………………………………….………...i

Acknowledgements……………………………………………………….…..……......ii

List of abbreviations…………………………..………………………….. ….....…….iii

Table of contents..............................................................................................................v

List of figures...................................................................................................................x

List of Tables.................................................................................................................xiii

1 Chapter 1 General introduction ........................................................................... 1

1.1 Clostridium acetobutylicum ................................................................................ 2

1.2 The history of acetone-butanol-ethanol (ABE) fermentation ............................. 2

1.3 Revival of ABE fermentation ............................................................................. 4

1.4 Biofuels ............................................................................................................... 6

1.5 The physiology and biochemistry of the ABE process ....................................... 7

1.6 The carbohydrate phosphotransferase system (PTS) ........................................ 11

1.7 Elements of Carbon Catabolite Repression (CCR) ........................................... 14

1.8 CCR in Gram negative bacteria ........................................................................ 15

1.9 CCR in Gram positive bacteria ......................................................................... 16

1.9.1 HPr and Crh ............................................................................................... 18

1.9.2 HPrK/P ....................................................................................................... 19

1.9.3 CcpA .......................................................................................................... 21

1.10 Elements of carbon catabolite repression in clostridia ..................................... 23

1.11 Fructose 1,6-bisphosphatase ............................................................................. 26

1.11.1 Fbpase class I ............................................................................................. 28

1.11.2 Fbpase class II (GlpX) ............................................................................... 28

1.11.2.1 In E. coli ............................................................................................. 28

1.11.2.2 In Corynebacterium glutamicum ....................................................... 29

1.11.2.3 In Mycobacterium tuberculosis ......................................................... 29

1.11.2.4 In Bacillus species ............................................................................. 30

1.11.3 Fbpase class III .......................................................................................... 30

1.11.4 Fbpase class IV and V ............................................................................... 30

1.12 Project aim ........................................................................................................ 32

2 Chapter 2 General materials and methods ....................................................... 33

2.1 Bacterial strains, growth conditions, primer and vector details ........................ 34

2.1.1 E. coli strain growth conditions ................................................................. 36

vi

2.1.2 Primer sequences ....................................................................................... 38

2.1.3 Growth conditions for C. acetobutylicum ATCC 824 ............................... 39

2.2 Chromosomal DNA extraction from C. acetobutylicum ATCC 824 ................ 39

2.3 Polymerase Chain Reaction (PCR) for TOPO™ TA cloning .......................... 40

2.4 Gel electrophoresis............................................................................................ 40

2.5 TOPO™ cloning procedure .............................................................................. 40

2.6 Transformation of chemically competent E. coli .............................................. 41

2.7 Screening of clones ........................................................................................... 41

2.7.1 PCR analysis .............................................................................................. 41

2.7.2 Plasmid purification ................................................................................... 42

2.7.3 DNA Sequencing ....................................................................................... 42

2.7.4 Restriction analysis .................................................................................... 43

2.7.5 Transformation of E. coli mutant strains ................................................... 43

2.7.6 Fructose 1,6-bisphosphatase assay ............................................................ 45

2.7.6.1 Microbuiret protien assay .................................................................. 46

2.8 Cloning of the glpX and fbp genes in the Gateway expression system ............ 48

2.8.1 BP cloning reaction .................................................................................... 48

2.8.2 Transformation of chemically competent cells .......................................... 49

2.8.3 LR reaction ................................................................................................ 49

2.8.4 Transformation into expression host .......................................................... 49

2.9 Cloning of glpX and fbp by Ligation-Independent Cloning (LIC) ................... 50

2.9.1 LIC cloning reaction .................................................................................. 50

2.9.2 Purification and precipitation of PCR product .......................................... 51

2.9.3 T4 DNA Polymerase treatment of the purified PCR product .................... 52

2.9.4 Transformation into the cloning strain ....................................................... 52

2.9.5 Transformation into the expression strain ................................................. 53

2.9.6 PCR Screening ........................................................................................... 53

2.9.7 Expression of the Fbp and GlpX proteins .................................................. 53

2.9.8 Purification of protien under native conditions using immobilizing

metal-affinity chromatography (IMAC) .................................................... 54

2.9.9 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

(SDS-PAGE) .............................................................................................. 55

2.9.10 Dialyzing of the recombinant proteins ....................................................... 56

2.9.11 Cleaving the fusion protein with recombinant enterokinase ..................... 57

2.10 Growth of C. acetobutylicum ATCC 842 for RNA extraction ......................... 57

vii

2.10.1 RNA extraction for C. acetobutylicum ATCC 824 .................................... 58

2.10.2 Analysis of purified RNA .......................................................................... 58

2.10.3 Reverse transcriptase PCR ......................................................................... 59

2.11 Proteomic analysis ............................................................................................ 61

2.12 Bioinformatics analysis ..................................................................................... 61

3 Chapter 3 Characterisation of glpX gene .......................................................... 62

3.1 Introduction ....................................................................................................... 63

3.2 Results ............................................................................................................... 63

3.2.1 Bioinformatic analysis ............................................................................... 63

3.2.2 The cac1088 gene ...................................................................................... 65

3.2.3 The cac1572 gene ...................................................................................... 69

3.3 Cloning glpX and fbp genes using TOPO cloning system ................................ 74

3.3.1 Cloning of the C. acetobutylicum class II glpX gene ................................ 74

3.3.2 Determination of the orientation of the insertion using PCR

analysis ....................................................................................................... 77

3.3.3 Growth of E. coli strains on different selective media for

phenotype studies. ...................................................................................... 83

3.3.4 Chemical transformation and complementation of different E. coli

mutants. ...................................................................................................... 85

3.3.5 Fructose bisphosphatase assay ................................................................... 90

3.3.6 Analysis of sequenced recombinant plasmid ............................................. 92

3.4 Cloning fbp gene using TOPO cloning system. ................................................ 93

3.5 Discussion ......................................................................................................... 97

3.5.1 Cloning of glpX gene using TOPO cloning system ................................... 97

3.5.2 Cloning of fbp gene using TOPO cloning system ................................... 103

3.6 Conclusion ...................................................................................................... 104

4 Chapter 4 GlpX and Fbp overexpression and purification .......................... 105

4.1 Gateway cloning system ................................................................................. 106

4.1.1 Introduction .............................................................................................. 106

4.1.3 Results ...................................................................................................... 108

4.1.3.1 Cloning of the glpX gene into the donor vector pDONR™221....... 108

4.1.3.2 Transferring the glpX gene into the expression vector pET-60-DEST

..........................................................................................................111

4.1.3.3 Cloning of the fbp gene into the donor vector pDONR™

221 .......... 113

viii

4.2 Cloning and expression of the glpX and fbp genes using Ligation

Independent Cloning system (LIC) ................................................................. 116

4.2.1 Introduction .............................................................................................. 116

4.2.2 Results ...................................................................................................... 117

4.2.2.1 Amplification and purification of the glpX and fbp PCR products.. 117

4.2.2.2 Cloning the glpX into the pET-41 Ek/LIC vector ............................ 122

4.2.2.3 Expression of the glpX gene in the recombinant pET-41 Ek/LIC

vector........ ..........................................................................................................124

4.2.2.4 Cleavage of the recombinant fusion GlpX protein .......................... 127

4.2.2.5 Assay of the fructose 1,6 biphosphatase activity of the purified

recombinant GlpX protein.................................................................................. 128

4.2.2.6 Overexpression of the glpX gene in the mutant JB108 .................... 128

4.2.2.7 Complementation of JB108 mutant ................................................. 130

4.2.2.8 Assay the fructose 1,6-biphosphatase activity of the recombinant

GlpX from JB1081/pNova-glpX-3 crude extract. .............................................. 131

4.2.2.9 Cloning and expression of the fbp gene ........................................... 133

4.2.2.10 Expression of the fbp gene in the recombinant pET-41 Ek/LIC

vector.................................................................................................................. 136

4.2.2.11 Expression of the fbp gene in the mutant JB108 ............................. 138

4.2.2.12 Complementation of JB108 mutant via fbp gene ............................ 140

4.2.2.13 Assay of the fructose 1,6 biphosphatase activity of the recombinant

Fbp protein from a crude extract of the JB1083/ pNova-fbp-3.......................... 141

4.2.2.14 Comparision of the FBPase activities of recombinant GlpX (native

and tagged) and recombinant Fbp proteins ........................................................ 143

4.3 Discussion ....................................................................................................... 146

4.3.1 Overexpression of GlpX and Fbp proteins by Gateway cloning

system. ..................................................................................................... 146

4.3.2 Overexpression of GlpX and Fbp proteins by LIC system ...................... 147

4.3.3 Assay of the purified Fbp and GlpX recombinant fusion proteins .......... 150

4.4 Conclusion ...................................................................................................... 151

5 Chapter 5 Transcriptional analysis ................................................................. 152

5.1 Reverse transcriptase- Polymerase chain reaction (RT-PCR) ........................ 153

ix

5.1.1 Introduction .............................................................................................. 153

5.1.2 Results ...................................................................................................... 153

5.1.2.1 Determination the expression of glpX and hprK ............................. 153

5.1.2.2 QIAGEN® One-Step RT-PCR Kit ................................................... 158

5.1.2.3 One Step RT-PCR Master Mix Kit from Novagen®

....................... 160

5.2 Discussion ....................................................................................................... 163

5.3 Conclusion ...................................................................................................... 165

6 Chapter 6 General discussion .......................................................................... 166

6.1 The importance of intracellular FBP levels in CCR and strain

improvements .................................................................................................. 167

6.2 Conclusions and future work .......................................................................... 171

7 Appendix ........................................................................................................ 172

7.1 Sequence of the recombinant fusion tags ........................................................ 173

7.2 Bioline Hyperladders ...................................................................................... 174

7.3 HyperPAGE Prestained Protein Marker ......................................................... 175

7.4 pCR 2.1 TOPO Cloning Vector ...................................................................... 176

7.5 The donor vector pDONR™221. .................................................................... 177

7.6 The Gateway®

Nova pET-60-DEST™ vector ................................................ 178

7.7 The pET-41 Ek/LIC vector ............................................................................. 179

7.8 Amino acids abbreviation ............................................................................... 180

7.9 Calibration curve of protein ............................................................................ 181

7.10 FBPase enzyme assay curve ........................................................................... 182

7.11 Proteomic analysis of FBPase class II (GlpX) protein ................................... 183

8 References ...................................................................................................... 184

x

List of Figures

Figure 1.1: Biochemical Pathways in C. acetobutylicum ................................................ 8

Figure 1.2: The Phosphoenolpyruvate dependent phosphotransferase system ............... 12

Figure 1.3: Model of catabolite activation or repression in B. subtilis. .......................... 17

Figure 1.4: The model of the phosphotransferase system (PTS) mediated by the

catabolite repression in C. acetobutylicum based on B. subtils model. ........ 24

Figure 1.5: Pathway of gluconeogenesis. Adapted from Berg et al., (2002). ................ 27

Figure 2.1: Schematic illustration of a protein with a His•Tag attached at the N-terminal

binding to a metal-chelated affinity support (NTA-Ni). .............................. 54

Figure 3.1: Unrooted phylogenetic tree of characterised FBPases and the two putative

clostridial FBPases ....................................................................................... 64

Figure 3.2: Alignment of the putative C. acetobutylicum fructose 1,6-bisphosphatase II

(cac1088) sequence with the closest homologues. ....................................... 68

Figure 3.3: Multiple alignment of the putative C. acetobutylicum fructose 1,6-

bisphosphatase (cac1572) sequence with the top 10 results from the BLAST

search. ........................................................................................................... 73

Figure 3.4: Two agarose gels of PCR amplification and screeing of glpX gene. .......... 76

Figure 3.5: Illustration of the strategy to determine the orientation of the insert in the

pCR2.1-TOPO vector. .................................................................................. 78

Figure 3.6: Agarose gel of PCR analysis to to determine the orientation of the glpX gene

in the vector. ................................................................................................. 79

Figure 3.7: Illustration of the strategy to determine the orientation of the insert in the

vector by restriction enzymes. ...................................................................... 81

Figure 3.8: Agarose gel showing HindIII (A) and EcoRI (B) digests of purified plasmid.

...................................................................................................................... 82

Figure 3.9: Growth of E. coli strains on M9 minimal medium containing 0.4% glycerol.

...................................................................................................................... 84

Figure 3.10: Growth of JB108/pM1W strains on two M9 glycerol plates. ..................... 86

Figure 3.11: Growth of JB108/pM1W strains on two LB plates. .................................... 87

Figure 3.13: Growth of JB103/pM1W and JLD2402/pM1B on M9 glycerol plate. ........ 89

Figure 3.14: Growth of JB103/ M1B on M9 glycerol plate (+Tet). ................................ 89

Figure 3.14: Two agarose gels of PCR amplification and purification of fbp gene........ 96

Figure 4.1: Illustration of the two BP and LR reactions of Gateway® cloning

Technology. ................................................................................................ 107

xi

Figure 4.2: Agarose gel of PCR screening of the glpX gene in MAX-glpX isolates. ... 110

Figure 4.3: Agarose gel of the purified plasmids (pMAX-glpX). ................................. 110

Figure 4.4: Agarose gel of PCR screening of the glpX gene in Rosetta-glpX isolates. 111

Figure 4.5: Agarose gel of PCR screening of the fbp gene in MAX- fbp isolates. ....... 115

Figure 4.6: Agarose gel of the purified plasmids (pMAX-fbp). ................................... 115

Figure 4.7: Diagram of the Ek/LIC strategy. ................................................................ 116

Figure 4.8: Agarose gel of PCR amplification of the glpX and fbp genes. ................... 117

Figure 4.9: Agarose gel of purified PCR products of the glpX and fbp genes using

agarose gel purification (Ultrafree-DA). .................................................... 120

Figure 4.10: Agarose gel of purified PCR products of the glpX and fbp genes following

chloroform extraction and ethanol precipitation. ....................................... 121

Figure 4.11: Agarose gel of PCR screening of the glpX gene in Nova-glpX isolates. .. 122

Figure 4.12: Agarose gel of the purified pNova-glpX plasmids. .................................. 123

Figure 4.13: Agarose gel of PCR screening of the glpX after purification from Nova-

glpX-3 and Nova- glpX-4 isolates. ............................................................. 124

Figure 4.14: Agarose gel of PCR sreening of the glpX gene in BL21/ pNova-glpX-3

isolates. ....................................................................................................... 125

Figure 4.15: SDS-PAGE of purified GlpX proteins in the eluted fractions after His-tag

purification under native conditions. .......................................................... 126

Figure 4.16: Illustration of GlpX protein attached to tagged proteins at the N- and C-

terminus. Enterokinase cleavage site is shown by the horizontal arrow. .. 127

Figure 4.17: Agarose gel of PCR screening of the glpX gene in the JB108/ pNova-glpX-

3 isolates. .................................................................................................... 128

Figure 4.18: SDS-PAGE of purified GlpX proteins in the eluted fractions after His-tag


Figure 4.19: Growth of JB1081/pNova-glpX-3 and JB1082/pNova-glpX-3 strain on two

M9 glycerol plates. ..................................................................................... 130

Figure 4.20: Agarose gel of PCR screening of the fbp gene in Nova-fbp isolates. ....... 134

Figure 4.21: Agarose gel of the purified pNova-fbp plasmids. ..................................... 134

Figure 4.22: Agarose gel of PCR screeing of the fbp gene after purification. .............. 135

Figure 4.23: Agarose gel of PCR screening of the fbp gene in BL21/pNova-fbp-3

isolates. ....................................................................................................... 136

Figure 4.24: SDS-PAGE of the purified Fbp proteins in the eluate fractions after His-tag


xii

Figure 4.25: Agarose gel of PCR screening of the fbp gene in JB1083/pNova-fbp-3 and

JB1084/pNova-fbp-3 isolates. ..................................................................... 138

Figure 4.26: SDS-PAGE of the purified Fbp proteins in the eluate fractions after His-tag


Figure 4.27: Growth of JB1083/pNova-fbp-3 and JB1084/pNova-fbp-3 strains on two

M9 glycerol plates. ..................................................................................... 140

Figure 5.1: Illustration of glpX, hprK and 5-formyltetrahydrofolate cyclo-ligase gene

arrangement. ............................................................................................... 156

Figure 5.2: Agarose gel electrophoresis of PCR amplification of fragments between

hprK, glpX and a gene that encodes for 5-formyltetrahydrofolate cyclo-

ligase........................................................................................................... 157

Figure 5.3: Agarose gel of purified total RNA from a culture of C. acetobutylicum

grown in glucose minimal medium. ........................................................... 158

Figure 5.4: Agarose gel electrophoresis of RT-PCR attempt to amplify fragments

between hprK, glpX and a gene that encodes for 5-formyltetrahydrofolate

cyclo-ligase................................................................................................. 159

Figure 5.5: Agarose gel electrophoresis of a control RT-PCR reaction from the

Novagen® One Step RT-PCR Master Mix Kit. .......................................... 161

Figure 5.6: Agarose gel electrophoresis of RT-PCR reaction to amplify fragments

between hprK, glpX and a gene that encodes for 5-formyltetrahydrofolate

cyclo-ligase................................................................................................. 162

xiii

List of Tables

Table 2.1: Details of E. coli strains used in this study and their genotypes .................... 35

Table 2.2: Plasmids used in this study ............................................................................ 36

Table 2.3: Growth media composition ............................................................................ 36

Table 2.4: List of antibiotics ........................................................................................... 37

Table 2.5: Primer details ................................................................................................. 38

Table 2.6: Composition of the restriction digest mixture ............................................... 43

Table 2.7: The composition of the fructose bisphosphatase assay reaction mixture ...... 46

Table 2.8: Different amounts of BSA and dH2O that were needed in the 1 ml assay

mixture. ........................................................................................................... 47

Table 2.9: Hot start DNA polymerase reaction setup. .................................................... 50

Table 2.10: Temperatures and times that were used for amplification by KOD Hot Start

DNA Polymerase. ........................................................................................... 51

Table 2.11: T4 DNA polymerase reaction setup. ............................................................ 52

Table 2.12: Reaction components for small scale optimization incubated in room or

37°C temperature. ........................................................................................... 57

Table 2.13: Reaction mix for one-step RT-PCR ............................................................. 59

Table 2.14: RT-PCR Cycling steps (QIAGEN® One-Step RT-PCR Kit) ....................... 60

Table 2.15: Reaction mix for one-step RT-PCR ............................................................. 60

Table 2.16: RT-PCR Cycling steps (One Step RT-PCR Master Mix Kit Novagen®

) .... 61

Table 3.1: BLAST homology results for the deduced amino acid sequence of the

putative fructose 1,6-bisphosphatase II (cac1088). ....................................... 66

Table 3.2 BLAST homology results for the deduced amino acid sequence of the

putative fructose 1,6-bisphosphatase III (cac1572). ....................................... 70

Table 3.3: Locations of TOPOglpX primers on the sequence of cac1088 (glpX gene). . 75

Table 3.4: Growth of the E.coli mutants on LB, glycerol and their phynotype. ............. 84

Table 3.5: Growth of different E. coli mutant strains in LB and glycerol medium

supplemented with or without IPTG. ............................................................. 87

Table 3.6: The effect of different metabolites (effectors) on the reaction rate of the crude

GlpX enzyme compare to the control (no effector). ....................................... 91

Table 3.7: Locations of TOPOfbp primers on the sequence of cac1572 (fbp gene). ...... 94

Table 3.8: Comparision of the effect of various metabolites on the activity of different

GlpX enzymes. ............................................................................................. 102

xiv

Table 4.1: Locations of GATEglpX primers on the sequence of cac1088 (glpX gene).

...................................................................................................................... 109

Table 4.2: Locations of GATEfbp primers on the sequence of cac1572 (fbp gene). .... 114

Table 4.3: Locations of LICglpX primers on the sequence of cac1088 (glpX gene). ... 118

Table 4.4: Locations of LICfbp primers on the sequence of cac1572 (fbp gene). ........ 119

Table 4.5: The effect of different metabolites (effectors) on the reaction rate of the crude

recombinant GlpX enzyme compare to the control (no effector). ................ 132

Table 4.6: The effect of different metabolites on the reaction rate of the recombinant

Fbp enzyme compare to the control (no effector). ....................................... 142

Table 4.7: Comparisons of different effectors on the FBPase activity of GlpX (native

and tagged) and Fbp tagged of C. acetobutylicum. ...................................... 143

Table 4.8: Comparisons of different divalent cations require for FBPase activity of

GlpX (native and tagged) and Fbp tagged of C. acetobutylicum. ................ 144

Table 5.1: Locations of RNAglpX primers on the sequences of cac1088-cac1089 (glpX-

hprK genes). ................................................................................................. 154

Table 5.2: Locations of RNAhprK primers on the sequences of cac1089-cac1090 (hprK-

5-formyltetrahydrofolate cyclo-ligase genes). .............................................. 155

1

Chapter 1:

1 General introduction

General introduction

2

1.1 Clostridium acetobutylicum

The taxonomy of the Clostridium acetobutylicum is Bacteria; Firmicutes; Clostridia;

Clostridiales; Clostridiaceae; Clostridium. It is a rod-shaped, Gram positive, obligate

anaerobe that cannot grow in the presence of oxygen, and is capable of producing

endospores under environmental stress conditions (Vos et al., 2009). C. acetobutylicum

belongs to a large genus that includes several important pathogens such as C. botulinum

which produces the most neurotoxin proteins that cause botulism illness, C. perfringens,

C. difficile, and C. tetani (Montecucco and Molgó, 2005). Also within the same genus

is C. thermocellum which is capable of converting cellulosic substrate into ethanol

(Ram and Seenayya, 2005). C. acetobutylicum was once known as the Weizmann

organism, named after chemist Chaim Weizmann who originally utilised the bacteria to

produce acetone, butanol and ethanol (ABE) in a large scale fermentation (Jones and

Woods, 1986). The strain C. acetobutylicum ATCC 824 was isolated from garden soil

in Connecticut in 1924, and has the ability to ferment a wide range of biomass. This

strain has now been shown to be related to the Weizmann strain (Nolling et al., 2001).

The genome of the solvent-producing bacterium C. acetobutylicum ATTC 824 consists

of a 4-Mbp chromosome and a megaplasmid (around 200 kbp in size) that carries the

genes involved in the production of solvent, hence, this plasmid was named pSOL1

(Nolling et al., 2001).

1.2 The history of acetone-butanol-ethanol (ABE) fermentation

The first butanol production from a microbial fermentation was reported in 1861 by

Louis Pasteur (Pasteur, 1862). The shortage of natural rubber in the early 20th

century

stimulated an interest in producing butanol due to synthetic rubber was needed in

manufacturing of automobile tyres (Gabriel, 1928). This problem stimulated the

research of many scientists - one of them the chemist Chaim Weizmann, who isolated

and studied many microorganisms between 1912 and 1914. One of the isolates was C.

acetobutylicum (Jones and Woods, 1986). The First World War changed the future of

the ABE fermentation. The smokeless gun powder (cordite) was required in large

quantities for the production of munitions, and vast quantities of acetone were required

for the manufacture process (Killeffer, 1927; Gabriel, 1928; Jones and Woods, 1986).

Also, the automobile industry needed a lacquer, and butanol was the best solvent for the

production of lacquer. The acetone production during World War II was needed again

for military use (Jones and Woods, 1986).


3

In 1945 one-tenth of the acetone and two-thirds of the butanol in the USA were still

produced in USA by fermentation (Rose, 1961). However due to many political,

economical and scientific reasons around the 1950’s the large scale fermentation for

solvent production began to decline.

Several of the major drawbacks that played an important role in the decline of the ABE

fermentation industry were firstly, the destructive problem of bacteriophage infection of

the bioreactors, which could cause the production of ABE to be cut by half for the year

(Jones and Woods, 1986). Secondly, solvent toxicity, especially butanol which is very

toxic for bacteria even at low concentrations (1-2% v/v). This resulted in low final

product concentrations, and subsequently high solvent recovery costs. Thirdly, the

growing cost of biomass substrates and finally, the ever expanding petrochemical

industry began to produce solvents at competitive prices compared to fermentatively

produced ABE (Hasting, 1971).


4

1.3 Revival of ABE fermentation

As a result of the dramatic price increase of petrochemicals, interest in the ABE

fermentation process has been revived, which has encouraged the development of new

technologies to produce fuels from cheap, secure and renewable resources. The USA

aims to increase the production of biofuels by three fold by 2025 (Hansen et al., 2005).

There has been much research conducted on many fronts (such as in clostridial genetics,

bioreactor technology and identification of cheap, renewable feedstocks) to enable the

ABE fermentation to once again become economically competitive against fossil fuel

(Jones and Woods, 1986). On the molecular front, many of the genomes of

solventogenic clostridia have been sequenced such as C. beijerinckii NCIMB 8052,

C. beijerinckii BA101, and C. acetobutylicum ATCC 824 (Nolling et al., 2001),

C. beijerinckii BA101, a mutant strain which is derived from C. beijerinckii NCIMB

8052, is a hyper-butanol producing strain, that can tolerate butanol better than the

parental strain, and also butanol production is increased by 100% (Qureshi and

Blaschek, 2001).

Ingenious bioreactor design, which can remove ABE during the fermentation, has

mitigated the problems caused by solvent toxicity (Nakas et al., 1983). Many substrates

have been identified which can replace molasses and grains, including, microalgae

Dunaliella which can be utilized, with the addition of 4% glycerol, by C. pasteurianum

with the production of 16 g/L of solvents (Nakas et al., 1983). Bioinformatics analysis

of C. acetobutylicum ATCC 824 identified 11 genes encoding for proteins that are

involved in cellulose utilization (Nolling et al., 2001). Whey, which is a by-product of

the dairy industry, can be used as an economical fermentation feedstock for solvent-

producing clostridia such as C. acetobutylicum (Qureshi and Maddox, 2005). Also,

uptake of lactose which is the major sugar in whey has been confirmed to be catalyzed

by a phosphotransferase system (PTS) (see section 1.6) (Yu et al., 2007). Crude

glycerol which is the by-product of the biodiesel industry can be used as an alternative

substrate. This low quality crude glycerol contains a mixture of contaminated glycerol,

water, methanol, free fatty acids, and both organic and inorganic salts (Thomson and He

2006).


5

Glycerol is produced in huge quantities during the production of biodiesel. 10 lbs of

crude glycerol is made from every 100 lbs of biodiesel generated from vegetable oils

and animal fats (Thompson and He, 2006). Due to the low price, crude glycerol is very

competitive with other sugars that are used in biofuel production by microbial

fermentation (Yazdani and Gonzalez, 2007). Up to date, there is no economical viable

use for the crude glycerol and it cannot be used in cosmetics and pharmaceutical

industries due to the purification process not being economical (Dobson et al., 2011).

However, in the USA the price of crude glycerol is constantly decreasing from US

$0.20 / lb in 2001 to US $0.01 / lb in 2006 which makes it in the future a most

promising, economically viable biological fermentation substrate (Dasari, 2007).

Around 50% of the production cost of biofuels is due to the cost of the fermentation

substrate, hence, if crude glycerol can be used the total production cost of the biofuel

would be lower (Willke and Vorlop, 2008; Dobson et al., 2011).

Several organisms are able to grow in glycerol, however, the best microorganisms that

able to grow in glycerol and produce a valuable product belong to the genus

Clostridium. Recent study indicates that a newly isolated Clostridium butyricum

AKR102a from soil was able to grow in low-quality crude glycerol and produce 1,3-

propanediol (1,3-PD) which can be used in cars as a coolant (Ringel et al., 2011). When

the strain was grown in crude glycerol, 76.2 g/L of 1,3-PD was produced making it the

best non-engineered strain able to produce a valuable product in such a large quantity

(Wilkens et al., 2011). It has also been reported that C. acetobutylicum was able to co-

utilize glucose and glycerol (Vasconcelos et al., 1993). Carlos et al, 2003 reported that

C. acetobutylicum can grow on crude glycerol in the presence of glucose and produce

the same amount of solvents as when grown on pure glycerol in the presence of glucose.

A continuous culture of C. acetobutylicum over 70 days was able to produce 0.34

mol/mol of butanol with overall butanol productivity of 0.42 g/L/h with no sign of

degeneration (loss of pSOL1 plasmid leads the cells to not produce solvents) (Carlos et

al, 2003). Biodiesel is produced in many EU countries, with Germany the largest

producer and consumer of this biofuel with the annual production exceeding 2.5 billion

liters. However, these countries have severe problems in disposal of the excessive crude

glycerol generated from this industry and also the disposal process is quite expensive

(Silva et al., 2009). Therefore, establishing a biobutanol industry together with

biodiesel leads not only to increase the profit, but also solves the disposal problem.


6

1.4 Biofuels

Biofuels are fuels produced from biomass (Demirbas, 2007). Biomass is a term which

can refer to any organic biological material, however in this context, biomass usually

refers to matter such as crops, wood and waste residues produced by farming, the food

and drink processing industry and so forth. This biomass can be used for the production

of renewable energy. Lignocellulose, which is the most abundant biomass on the Earth,

is mainly composed of three sugars, glucose, arabinose, and xylose (Xiao et al., 2011).

Crop waste and forestry residues are examples of lignocellulosic biomass which does

not compete with animal feedstock and food industry (Weber et al., 2010). Biomass

usually needs to be degraded into manageable mono or disaccharides before microbial

conversion into solvents or biofuels can occur, and thermochemical, enzymatic (or a

combination of the two) pretreatment is usually employed (Brodeur et al., 2011).

Biofuel is the most promising alternative energy that will achieve the aims of reducing

CO2 emissions and the dependence on fossil fuel, also production and the trade of

biofuel can create new jobs and support the economy of the developing countries

(Groom et al., 2008).

There are two main types of liquid biofuel used for transport fuel:

Biodiesel is derived from biological sources such as animal fats and vegetable

oil. It has low emissions and is readily biodegradable, compared with

petrodiesel. The term biodiesel was introduced by the National Soy Diesel

Development Board in USA during 1992. Biodiesel can be used in the current

diesel engine with moderate or no modification to it. However, there are some

problems associated with the use of biodiesel in the current diesel engine, for

example, high viscosity and gum formation which at the end damages the diesel

engine (Ramadhas et al., 2003).


7

Bioalcohols, for example bioethanol produced by yeasts, are already being used

in Brazil, the USA and some European countries on a large scale, and have

potential to be one of the leading renewable biofuels in the coming 20 years due

to the increase in crude oil price. Bioethanol is produced from sugar cane in

Brazil and from corn in USA, however this biomass stream can also be utilised

for human and animal nutrition, which causes these feedstocks to be subject to

erratic price fluctuations and become a target for the growing food versus fuel

movement (Hägerdal, 2006).

Biobutanol, which is butanol that is derived from biomass, is produced by the

solventogenic clostridia such as C. acetobutylicum. The World market for

butanol is currently 350 million gallons per year. The USA alone consumes 220

million gallons per year (Shapovalov and Ashkinazi, 2008). It is mainly used as

solvent and an intermediate in chemical production (Lee et al., 2008).

However, Butanol has several advantages as a transport fuel over ethanol.

Butanol has a higher energy content compared to ethanol, it blends better with

gasoline at any concentration, and it can be used in current cars without any

modification to the engine. It also evaporates six times less than ethanol, is not

corrosive compared to ethanol, hence, can be distributed by pipelines. (Dürre,

2007; Shapovalov and Ashkinazi, 2008).

1.5 The physiology and biochemistry of the ABE process

The solvent-producing clostridia have two main metabolic phases. The initial growth

phase - the acidogenic phase - produces hydrogen, carbon dioxide, acetate, and butyrate

(Figure 1.1). Production of the acids results in a decrease of the pH of the medium.

The second phase of the fermentation is the solventogenic phase, in which the acetate

and butyrate are taken up from the medium then converted into solvents, and this

subsequently results in an increase of the pH of the medium (Davies and Stephenson,

1941; Johnson et al., 1931; Reilly et al., 1920; Rose, 1961).

The build-up of large amounts of acetic and butyric acids puts the cell in danger by

dissipating the proton gradient across the cytoplasmic membrane. As a result, the cells

stop the formation of acids and shift towards the production of neutral solvents, the

acids being mostly reassimilated and converted to acetone, butanol and ethanol (Dürre,

2002).


8

Figure ‎1.1: Biochemical Pathways in C. acetobutylicum

Figure adapted from Jones & Woods, 1986. Reactions which predominate during the

acidogenic phase and the solventogenic phase of the fermentation are shown by thin

and thick arrows, respectively. Enzymes are indicated by letters as follows: (A)

glyceraldehyde 3-phosphate dehydrogenase; (B) pyruvate-ferredoxin oxidoreductase;

(C) NADH-ferredoxin oxidoreductase; (D) NADPH-ferredoxin oxidoreductase; (E)

NADH-rubredoxin oxidoreductase; (F) hydrogenase; (G) phosphate acetyltransferase

(phosphotransacetylase);


9

(H) acetatekinase; (I) thiolase (acetyl-CoA acetyltransferase); (J) 3-hydroxybutyryl-

CoA dehydrogenase; (K) crotonase; (L) butyryl-CoAdehydrogenase;

(M)phosphatebutyltransferase (phosphotransbutyrylase); (N) butyrate kinase; (O)

acetaldehyde dehydrogenase; (P) ethanol dehydrogenase; (Q) butyraldehyde

dehydrogenase; (R) butanol dehydrogenase; (S) acetoacetyl-CoA:acetate/butyrate:CoA

transferase; (T) acetoacetate decarboxylase; (U) phosphoglucomutase; (V) ADP-

glucose pyrophosphorylase; (W) granulose (glycogen) synthase; (X) granulose

phosphorylase.

Under certain conditions C. acetobutylicum can convert pyruvate into lactate. Lactic

acid is not produced under normal conditions, but this occurs when the hydrogenase

activity is inhibited by carbon monoxide or the microbe runs out of iron (Jones and

Woods, 1986). This pathway only operates as an alternative pathway in order to allow

the continuation of NADH oxidation and energy generation. In the acidogenic pathway,

butyrate and acetate are produced from butyryl-CoA and acetyl-CoA which results in

the production of ATP at end of the pathway.

There are four main enzymes responsible for the formation of acids; phosphate

acetyltransferase, and acetate kinase, encoded by pta and ack genes, for the production

of acetic acid, and phosphate butyryl-transferase and butyrate kinase encoded by ptb

and buk genes for the production of butyrate (Jones and Woods, 1986). In the

acidogenic phase the highest amount of hydrogen production occurs, concurrent with a

high rate of glucose consumption. The first decrease of hydrogen production is

associated with a reduction in the metabolic activity and cell growth in the culture.

Thus in this stage, the decrease in the hydrogen production is due to the reduction in the

metabolic rate rather than inhibition of hydrogenase activity (Kim and Zeikus, 1985).

The start of the solventogenic phase coincides with a fall in the pH of the medium

which is linked to accumulation of acids. The addition of butyrate and acetate to a

culture of C. acetobutylicum at pH 5.0 was reported to accelerate the induction of

solventogenesis (Bernhauer and Kurschner, 1935; Reilly et al., 1920), which was

followed by a decrease in the growth rate and hydrogen production. The acetate and

butyrate uptake only occurs when the sugars in the medium have been depleted.


10

The genes that are required for solvent production in C. acetobutylicum are aad, ctfA,

ctfB, and adc which are located on a 192 kbp megaplasmid pSOL1. C. acetobutylicum

may lose pSOL1 resulting in the loss of the ability to produce solvents. Strains that

have lost the plasmid are said to have degenerated (Scotcher and Bennett, 2005).

Solvent production serves as a detoxification process in response to the accumulation of

acids which result in conditions that reduce the cells growth rate (Hartmanis et al.,

1984). The shift from acidogenic phase to solventogenic phase is accompanied by a

change in the production ratios of hydrogen and CO2 (a decrease in hydrogen

production and an increase in CO2 production. In the solventogenic phase, the cell

metabolism continues until the concentration of solvents reaches an inhibitory point

which is usually around 20 g/L. The most toxic solvent is butanol, and the solvent

production is inhibited when the concentration of butanol reaches 13 g/L. Recent studies

have confirmed that growth is completely inhibited by the addition of 12 to 16 g/L of

butanol. However, the addition of ethanol and acetone to the medium reduces the

growth by 50% at a concentration of 40 g/L and growth was totally inhibited at a

concentration of 70 g of acetone and 50 g to 60 g of ethanol per litre (Leung and Wang,

1981; Jeanine and Antonio, 1983).

Butanol toxicity is related to the hydrophobic property of this molecule. The main effect

of this compound appears to be the disruption of the phospholipid bilayer of the cell

membrane which results in an increase in membrane fluidity. Furthermore, a recent

study has observed an increase in the ratio of saturated to unsaturated fatty acids in the

membranes both from solventogenic phase cells and vegetative cells grown in the

presence of butanol (Schneck et al., 1984). Hence, this increase in the saturated fatty

acids in the membrane is due to the physiological response of the cell to tolerate the

effect of increased membrane fluidity, which is similar to the response of cells grown at

high temperature. Butanol tolerance could be enhanced by the addition of saturated

fatty acids to the medium which results in an increase in the content of saturated fatty

acids in the membrane. Also, a decrease in fermentation temperature may increase the

butanol tolerance during the solventogenic phase (Rogers, 1984).


11

1.6 The carbohydrate phosphotransferase system (PTS)

Transport from the environment to inside the cell is the first step in any sugar

metabolism pathway and the PTS system is the main sugar transport system in many

Gram positive and Gram negative microorganisms (Postma et al., 1993; Deutscher et

al., 2006). In 1964, this system was firstly reported in E. coli by Kundig et al. (1964).

The PTS is a predominant transport system in many facultative and obligate anaerobic

bacteria and involves the uptake and phosphorylation of a large number of carbon

sources. Although its main function is as a transport system, it can also be involved in

movement towards carbon sources (chemotaxis), and the regulation of gene expression

(Deutscher et al., 2006; Postma et al., 1993). All the PTS systems that have been

reported catalyze the following process:

P-enolpyruvate (inside cell) + carbohydrate (outside cell)

pyruvate (inside cell) +

carbohydrate-P (inside cell).

Sugar uptake in clostridia relies heavily but not completely on the

phosphoenolpyruvate-dependent PTS (Figure 1.2). The main mechanism of

carbohydrate transport in C. acetobutylicum and C. beijerinckii is the PTS (Mitchell and

Tangney, 2005). Many sugars such as sucrose, maltose, glucose, lactose and fructose

have been reported to be PTS substrates in C. acetobutylicum and the genes of these

systems have been characterised (Tangney and Mitchell, 2000; Tangney et al., 2001;

Tangney and Mitchell, 2005; Yu et al., 2007). As in many bacteria, the expression of

PTS genes is induced by their substrates and they are subject to carbon catabolite

repression (CCR) by glucose. Neither sucrose nor maltose can be utilized by the

growing bacteria when cultured in the presence of glucose (Tangney and Mitchell,

2007).

PTS


12

Figure ‎1.2: The Phosphoenolpyruvate dependent phosphotransferase system

Organisation of the phosphoenolpyruvate dependent phosphoTransferase system

(PEP: PTS). Figure adapted from Mitchell and Tangney, 2005. Enzyme I and heat

stable histidine-phosphorylatable protein (HPr) are the general PTS proteins for all

PTS’s. The substrate specific enzyme II can have highly variable domain structure,

present as a single polypeptide or individual proteins. A phosphate (~p) is donated

from PEP to enzyme I, HPr, enzyme II, and then the sugar.

Sugar~P

~P

HPr

Enzyme I

Sugar

PEP

~P

~P

~P

~P

(His-15)

E IIAE IIB

E IIC

Membrane


13

The PTS is composed of a multiprotein phosphoryl transfer chain. The first two

proteins, enzyme I (EI) and HPr are soluble proteins that are involved in the

phosphorylation of all of the PTS carbohydrates in any microorganism; hence, they

have been called the general PTS proteins (Deutscher et al., 2006; Tangney and

Mitchell, 2005; Postma et al., 1993). Bioinformatics analysis has revealed that most of

the EIs from Gram-positive and Gram-negative bacteria show strong identity and

consist of around 570 amino acids with size about 63 kDa (Kuang-Yu and Saier Jr,

2002). EI is phosphorylated in the presences of Mg2+

at a conserved histidine site which

is located in the N-terminal region (His-189) in E. coli (Weigel et al., 1982). Moreover,

several studies reported Mg2+

is needed for EI phosphorylation, but is not needed for

transfer of the phosphate from EI to the conserved His-15 in HPr (Weigel et al., 1982;

Chauvin et al., 1996). The C terminal of the enzyme which contains the PEP binding

site is needed for dimerization of EI. Only dimer can be phosphorylated by PEP, which

may imply that monomer to dimer transition is involved in regulation of the enzyme

activity (Chauvin et al., 1996).

HPr, which is the heat stable histidine-phosphorylatable protein and EI are encoded by

the genes ptsH and ptsI, respectively. The other PTS proteins are the substrate-specific

enzyme II (EII) domains, which might exist separately or connected within a single

polypeptide chain (Tangney and Mitchell, 2007). These domains are referred to as the

EIIA, EIIB, and EIIC, whereas a small number of systems were reported to have an

extra EIID domain such as the fructose PTS system in B. subtilis (Martin-Verstraete et

al., 1990; and Reizer, 1992). The first two domains IIA and IIB are hydrophilic and

both contain sites for phosphorylation, whereas IIC and IID are membrane bound

proteins and their function is to translocate the substrate. The EIIC domain is more

hydrophobic than the IID (Saier Jr and Reizer, 1992; Postma et al., 1993).

The clostridial system consists of functional proteins equivalent to those in other

bacteria. Hence, the soluble part of the C. beijernckii NCIMB 8052 PTS complemented

the membranes prepared from Bacillus subtilis, C. pasteurainum and E. coli for glucose

phosphorylation (Tangney and Mitchell, 2005).


14

Also, clostridial extracts restored the PTS activity of the extracts prepared from ptsI and

ptsH mutants of B. subtilis, Staphylococcus aureus and E. coli (Saier and Reizer, 1992;

Tangney and Mitchell, 2005). The phosphotransferase system (PTS) has been show to

play a main role in bacterial CCR through HPr (Zeng and Burne, 2010; Deutscher et al.,

2006).

1.7 Elements of Carbon Catabolite Repression (CCR)

More than a century ago, it was reported that growth on glucose can decrease the

activity of specific enzymes in yeast and bacteria. This event became known as the

glucose effect. The first description of this phenomenon was by Dienert in 1900 in

France. He reported that Saccharomyces cerevisiae cells that had been adapted to

galactose, lost this adaptation directly after they were exposed to glucose or fructose

(Dienert, 1900). Another study was done in Wageningen, Netherlands by Sohngen and

Coolhaas, who measured the influence of temperature on the glucose effect in S.

cerevisiae (Sohngen and Coolhaas, 1924). At the beginning of the 1940s, Jacques

Monod studied B. subtilis and E. coli and concluded that “in each organism, a specific

hierarchy existed for the utilization of carbon sources, with glucose usually being in the

top of it” (Monod, 1942). Later, it was found that the preferred carbon sources such as

glucose, fructose and sucrose could repress the synthesis of the enzymes that are needed

for the transport and metabolism of less favourable carbon sources. This observable

fact became known as carbon catabolite repression (CCR) (Deutscher et al., 2006).

When the preferred sugar in a culture is depleted, cells begin to synthesize the necessary

enzymes for the transport and metabolism of the less favourable sugar, and usually

show a lag phase before they can resume growth again. Therefore, the glucose effect

phenomenon reported in the 1900s can be considered as carbon catabolite repression.

Carbon catabolite repression can be defined as the inhibitory effect of a particular sugar

in the growth medium on gene expression or the activity of enzymes involved in the

catabolism of other sugars (Deutscher et al., 2006). CCR can be mediated by various

mechanisms, which can repress the transcription of catabolic genes via specific or

global regulators (Deutscher, 2008). The main CCR mechanism in firmicutes is

mediated by the global regulator catabolite control protein A (CcpA) as will be

explained later in greater detail.


15

1.8 CCR in Gram negative bacteria

A major global regulator in E. coli is the cyclic AMP receptor protein (CRP), also

called the catabolite activator protein (CAP) which is a homodimeric transcription

protein with each monomer composed of two functional domains. The first domain is

located in the N-terminal region which contains a cAMP binding site and the second

domain is located in the C-terminal which contains a specific DNA binding site (Cheng

and Lee, 1998; Busby and Ebright, 1999; Li and Lee, 2011). CRP activates

transcription at many promoters in the presence of cAMP by binding to specific

palindromic sequence of 22 bp located in or upstream of the promoter (Busby and

Ebright, 1999; Lawson et al., 2004). In absence of glucose, cAMP is present at a high

concentration which stimulates CRP binding to the specific CRP-site to activate

transcription. However, under glucose growth conditions, cAMP is present at a low

level, hence, no transcriptional activation occurs (Lawson et al., 2004; Crasnier-

Mednansky et al., 1997).

The catabolite repressor /activator (Cra) protein is another global regulator that mediates

the central metabolic pathways in Gram negative bacteria (Chavarría et al., 2011). Cra

was initially known as the fructose repressor (FruR) for repressing of the genes that are

involved in fructose metabolism, however, more studies reported that FruR is not

specific for repressing fructose utilizing genes but has a global regulator function which

can repress or activate many catabolic genes. Therefore, it was termed Cra (Ramseier et

al., 1993; Shimada et al., 2010; Chavarría et al., 2011). Cra belongs to the GalR-LacI

family of proteins which contain two domains, an N-terminal helix-turn-helix domain

which recognises a specific imperfect palindromic DNA sequence of around 12 bp and

binds to it, and a second C-terminal domain which is the inducer binding domain by

which fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate bind to the protein

(Saier Jr and Ramseier, 1996; Shimada et al., 2010).

Repression and activation of the catabolic genes are dependent on the location of the

Cra-binding sites. Therefore, activation occurs when the Cra-binding site is located

within or downstream of the promoter, but, gene repression occurs when the Cra-

binding site is located upstream of the promoter (Saier Jr and Ramseier, 1996).


16

Cra is regulated by FBP and/ or fructose 1-phosphate (effectors) via stimulating

dissociation of Cra from the Cra-binding sites. As a result, the genes that are associated

with a Cra-binding site located upstream of the promoter are activated by Cra and since

the effector binds to Cra, this leads to displacement of the protein from the Cra-binding

site, hence, these genes are subject to catabolite repression. Whereas, the genes are

associated with a Cra-binding site that is located within or downstream of the promoter

are repressed by Cra and when the effector binds to Cra, this leads to displacement of

the protein from the Cra-binding site, and hence these genes are subject to catabolite

activation (Saier Jr and Ramseier, 1996).

To date, it is unclear why the cell has two different global regulators, Cra and CRP, for

controlling carbon metabolism (Shimada et al., 2010). However, recent studies indicate

the crp gene encoding for CRP is under the control of the Cra regulator which makes it

the true global regulator in Gram negative bacteria (Shimada et al., 2010; Chavarría et

al., 2011).

1.9 CCR in Gram positive bacteria

Catabolite control protein A ( CcpA) is the the global regulator that controls the central

metabolic pathways in Gram positive bacteria (Singh et al., 2008). It also can regulate

genes that are involved in sporulation, toxin production and biofilm formation (Varga et

al., 2004; Varga et al., 2008; Antunes et al., 2010). Like Cra in Gram negative bacteria,

CcpA belongs to the GalR-LacI family (Shimada et al., 2010; Deutscher et al., 2008).

CCR occurs when HPr of the PTS is phosphorylated at a serine site (P-Ser-HPr) by HPr

kinase which forms a complex (P-Ser-HPr-CcpA) that binds to a specific DNA site in

the presence of FBP (Figure ‎1.3) (Deutscher et al., 2008). Moreover, B. subtilis

contains a homologous protein to HPr termed Crh which can be involved in CCR

(Figure ‎1.3). All these elements of CCR are explained below in greater detail.


17

Figure ‎1.3: Model of catabolite activation or repression in B. subtilis.

Figure adapted from Lorca et al., 2005. Proposed sensory transduction pathway by

which exogenous glucose is believed to activate the CcpA transcription factor to

promote catabolite repression (CR) or catabolite activation (CA) by binding to a

catabolite responsive element (cre) in the control region of a target gene. (i) Proteins

primarily involved are the glucose phosphotransferase system, including enzyme I,

HPr, and IIBCAGlc

, (ii) glycolytic enzymes, (iii) the ATP-dependent fructose 1,6-

bisphosphate (FBP)-dependent HPr/Crh kinase/phosphatase HprK, and the two HPr

and Crh protein targets of HprK that independently bind to CcpA to activate it for

binding to cre sites.


18

1.9.1 HPr and Crh

The general PTS protein HPr is the central processing unit in the regulation of carbon

metabolism in the low GC Gram positive bacteria. HPr is a small protein consisting of

about 90 amino acids only (9 to 10 kDa) (Postma et al., 1993). HPr can be

phosphorylated at either His-15 by enzyme I and PEP or Ser-46 by ATP. As a result,

four different forms exist; HPr (dephosphorylated form), HPr phosphorylated at either

His-15 (P-His-HPr) or Ser-46 (P-Ser-HPr), and the double phosphorylated HPr

(Deutscher et al., 2006). The active-site histidyl residue (His-15) in HPr which

participates in the PTS is located in the N-terminal region, and the region around the

histidyl residue is conserved in most bacteria (Deutscher et al., 1986; Deutscher et al.,

2006). In the B. subtilis sequencing project, a gene was identified that encodes a

protein that shares 45% sequence identity to HPr. It was therefore termed catabolite

repression HPr-like protein (Crh) (Martin-Verstraeter et al., 1999; Galinier and Haiech

et al., 1997). To date, Crh has been found only in bacillus spp. has the same regulatory

site (Ser-46) as HPr which is involved in carbon catabolite repression. However, His-15

that is involved in sugar transport is replaced by glutamine in Crh, thus it cannot

participate in sugar transport (Galinier et al., 1997; Schumacher et al., 2006).

Martin-Verstraete et al, (1999) reported that HPr can completely substitute for Crh

deletion in carbon catabolite repression of the lev operon which encodes for a fructose-

specific PTS and levanase that catalyse metabolism of fructose polymers. However,

Crh can only partly substitute for HPr deletion. However, specific function for Crh in

B. subtilis has been described by Warner et al, (2000). They reported that the Mg-

citrate transporter (CitM) of B. subtilis, which is the main citrate uptake transporter

during growth on citrate as the sole carbon source, is exclusively regulated by Crh

during growth on gluconeogenic substrates such as succinate and glutamate (Warner et

al., 2000). CitM transports citrate or isocitrate into the cell in complex with divalent

cations such as Mg, Zn, and Mn, hence it is termed the Mg-citrate transporter (Warner

et al., 2000).


19

1.9.2 HPrK/P

The metabolism of several carbon sources affects the activity of HPrK/P which is a

bifunctional protein kinase/ phosphorylase, that reacts to changes of the concentration of

FBP, ATP, pyrophosphate (PPi), and inorganic phosphate (Pi) (Hogema, et al., 1998).

The phosphorylation of HPr at Ser-46 requires the HPrK/P protein kinase and ATP

(Deutscher and Saier Jr, 1983). The substitution of Ser-46 with alanine, tyrosine, or

aspartate in B. subtilis HPr prevented the FBP-stimulated ATP-dependent

phosphorylation of HPr (Eisermann et al., 1988). Also, mutation at this site affected the

PEP-dependent phophorylation at His-15 (Reizer et al., 1989). FBP is needed to

stimulate HPrK/P to phosphorylate either HPr or Crh in B. subtilis (Jault et al., 2000).

HPrK/P kinase activity was identified in several Gram positive bacteria more than 28

years ago. However, the hprK gene was discovered only during the B. subtilis

sequencing project (Jault et al., 2000; Deutscher and Saier Jr, 1983). When the

concentrations of FBP and ATP increase, the kinase activity of HPrK/P is predominant;

on the other hand, the phosphorylase activity becomes predominant when the

concentration of Pi increases (Kravanja et al., 1999; Jault et al., 2000). HPrK/P can

dephosphorylate P-Ser-HPr in presence of inorganic phosphate producing PPi. The

dephosphorylation is stimulated by Pi, and due to P-Ser-HPr dephosphorylation being a

reversible mechanism, hence PPi can substitute for ATP as the phosphoryl donor for

HPr at high concetration (Figure ‎1.3) (Deutscher et al., 1985; Nessler et al., 2002;

Kravanja et al., 1999). Kravanja et al, (1999) reported that the kinase activity of

HPrK/P was inhibited at high concentration of Pi (50 mM) present under starvation

conditions, however, a drop in the concentraction of Pi (4 mM) was detected after

adding glucose to the medium which led to restoration of the kinase activity of HPrK/P.

Therefore, the HPrK/P protein plays a vital rule in adjusting the level of P-His-HPr and

P-Ser-HPr according to the nutritional state of the cell (Kravanja et al. 1999) (Figure

‎1.3).

Jault et al, (2000) showed that when the FBP concentration increases, HPrK/P was

strongly stimulated to phosphorylate HPr at Ser-46. Therefore, there is a correlation

between FBP concentration and HPrK/P stimulation. Also, it has been shown that FBP

(20 mM) stimulates the kinase activity of B. subtilis and C. acetobutylicum HPrK/P and

the kinase activity is stronger when low concentrations of ATP (0.4 mM) are used (Jault

et al., 2000; Tangney et al., 2003).


20

The intracellular concentrations of ATP, Pi, PPi and FBP are determined by whether the

bacteria are utilizing a favoured sugar or not (Deutscher et al., 2006). B. subtilis cells

grown on glucose were found to contain more FBP (14.1 mM) and PPi (6 mM) than

cells grown on less favourable substrate such as succinate which was found to contain

only 1.4 mM for FBP and 1.2 mM for PPi (Blencke et al., 2003; Poncet et al., 2004;

Singh et al., 2008). This explains why about 65% of the HPr in B. subtilis grown on

glucose is present as P-Ser-HPr and the rest is present as HPr, P-His-HPr, and the

doubly phosphorylated HPr (Singh et al., 2008). However, when succinate is the sole

carbon source, the majority of the protein is in the form of HPr, together with a small

amount of P-His-HPr. Therefore, the two antagonistic activities of HPrK/P and the

percentage of various form of HPr are regulated mainly by these metabolites (Moreno et

al., 2001; Singh et al., 2008). HPrK/P kinase activity in the presence of less than 1 mM

FBP was very low, however, higher FBP concentrations in the assay reaction mix led to

an increase in the HPrK/P kinase activity (Jault et al., 2000; Singh et al., 2008). A few

mM of FBP can overcome the inhibition of the kinase activity of HPrK/P excreted by

Pi, hence, the major physiological function of FBP may be to stop the inhibition of

HPrK/P by Pi (Kravanja et al. 1999; Brochu and Vadeboncoeur, 1999).

In the firmicutes, the hprK gene, which encodes the bifunctional enzyme HPr K/P, is

frequently the first within an operon, and the second gene encodes a prolipoprotein

diacylglycerol transferase (lgt) which may be involved in metabolism and membrane

anchoring. This association of these two genes may indicate that the functions of the

two enzymes are related but until now there is no experimental evidence to support the

assumption (Deutscher et al., 2006). However, in clostridia the lgt and hprK genes are

located in different places in the genome. In the case of C. acetobutylicum, hprK is

preceded by the glpX gene which appears to encode a fructose 1.6 bisphosphatase

(Tangney et al., 2003).

No Gram negative bacteria have been identified which possess the HPrK/P kinase, even

though their HPr proteins possess Ser-46 and share 45% overall sequence identity to

HPr`s from the Gram positive bacteria (Deutscher and Engelmann, 1984).


21

However, the difference around the Ser-46 region is responsible for the failure of

HPrK/P kinase from Gram positive organisms to phosphorylate the E. coli HPr

(Deutscher and Saier, 1983; Reizer et al., 1989). The replacement of the amino acids

next to Ser-46 in the Mycoplasma capricolum HPr with the equivalent amino acids of E.

coli, resulted in poor phosphorylation with ATP (Zhu et al., 1998).

1.9.3 CcpA

CcpA is the catabolite control protein A which belongs to the GalR–LacI regulatory

protein family as mentioned before (Deutscher et al., 2006). It has a helix-turn-helix at

the N-terminal which is responsible for DNA recognition and binding to a specific site

called cre (catabolite responsive element) which is found in the promoter region of

catabolic operons. The C-terminal of CcpA is engaged in the recognition of the

phosphoserine and binding to P-Ser-HPr complex in the presences of FBP (Jones et al.,

1997 and Loll et al., 2007). CcpA either by activation or repression is needed in order

to maximize the efficiency of energy and carbon metabolism in the cell (Fujita, 2009).

CcpA regulates the expression of around 400 genes which is about 10% of the B.

subtilis genome (Nessler et al., 2003). In B. subtilis, CcpA (global regulator) and XylR

(specific regulator) are required for full glucose repression of the xyl operon, however,

only CcpA is needed for full fructose repression (Dahl and Hillen, 1995). CcpA can

act as a repressor or activator depending on the location of the cis acting catabolite

responsive element (cre) which is a 14 bp palindromic sequence, reported by Weickert

and Chambliss (1990). The cre consensus sequence is: TGWAANC*GNTNWCA,

where the star signs (*) indicates the centre of the palindrome, W is adenine or thymine,

and N is any base (Weickert and Chambliss, 1990). Therefore, repression occurs when

the cre site is located within or downstream of the promoter, but, gene activation occurs

when the cre site is located upstream of the promoter (Henkin, 1996). In B. megaterium

and B. subtilis, the ccpA gene is expressed in a constitutive manner and this does not

depend on the growth conditions. Instead, CcpA is controlled at a post translational

level (Hueck et al., 1995; Henkin, 1996). Also, CcpA controls the efficiency and

optimizes the catabolism of glucose through repression of alternative metabolism

pathways (Van der Voort et al., 2008).


22

Moreno et al, (2001) reported that CcpA can mediate gene repression or activation in a

glucose independent manner. Computer analysis can identify the putative cre sites that

are involved in catabolite repression but fails to identify cre sites that are involved in

catabolite activation. CcpA binds to cre sites as a dimer in a tetrameric protein complex

in the presence of either co-factors HPr-P-Ser or Crh-P-Ser (Moreno et al., 2001).

The amounts of CCR elements such as HPrK/P, CcpA, HPr, and Crh in the cell are not

affected by the presence of different carbon sources (Singh et al., 2008). Fructose 1,6

bisphosphate is needed to stimulate the interaction between CcpA and HPr-P-Ser and is

needed to enhance binding of the CcpA-HPr-P-Ser complex to cre sites (Henkin, 1996;

Presecan-Siedel et al., 1999; Antunes et al., 2010). Kim et al, (2005) reported that

interaction between CcpA and RNA polymerase is needed in order to inhibit the

transcription of the amyE gene. Actually, a cre site (centered at +4.5) is overlapping

with the amyE promoter area, but CcpA would not prevent RNA polymerase from

binding to the promoter (Kim et al., 2005). Also CcpA cannot bind to cre sites in the

absence of HPr-P-Ser (Kim et al., 1998). Some operons have more than one cre site,

for example, the xyl operon has 3 cre sites. The major one is centered at +130.5, and

two supplementary sites are located at -35.5 and +219.5 (Kim et al., 2005). When

CcpA binds to the major cre site that is located downstream of the xyl operon

transcriptional start site, the CcpA would not behave as roadblock (inhibit the

elongation) instead, it inhibits the initiation of transcription. These results suggest that

CcpA has a regulatory function other than simply binding to cre sites (Blencke et al.,

2003; Lorca et al., 2005). Not all putative cre sites that have been identified have been

shown to be functional (Van der Voort et al., 2008; Miwa et al., 2000). Actually, most

of the genes that are activated by CcpA appear to lack a cre site and this indicates that

the catabolite repression mechanism is unable to elicit catabolite activation by CcpA

due to the absence of the cre sites (Blencke et al., 2003; Lorca, et al., 2005).


23

1.10 Elements of carbon catabolite repression in clostridia

The ptsH gene which encodes for HPr has been identified in the C. acetobutylicum

genome sequence, and when cloned into an E. coli ptsH mutant, the clostridial gene

complemented the mutation. The alignment of the HPr amino acid sequence of low GC

Gram positive bacteria with HPr of C. acetobutylicum showed that the sequence around

the His-15 site which participates in PTS transport was conserved (Tangney et al.,

2003), whereas the region around the Ser-46 site, which is involved in CCR and is well

conserved in HPr proteins from other Gram-positive bacteria, is not conserved in C.

acetobutylicum HPr (Tangney et al., 2003). This protein can, however, be

phosphorylated at Ser-46 by HPrK/P kinase in an ATP-dependent matter, and this

activity is stimulated by FBP (Figure 1.4). Moreover, the clostridial HPrK/P kinase can

phosphorylate the purified HPr from B. subtilis in the presence of FBP. Thus, it is

suggested that the carbon catabolite repression model of low GC Gram positive bacteria

also applies to C. acetobutylicum (Tangney et al., 2003; Tangney and Mitchell, 2005).


24

Figure ‎1.4: The model of the phosphotransferase system (PTS) mediated by the

catabolite repression in C. acetobutylicum based on B. subtils model.

The PTS is a transport system that has a vital role in CCR in low GC Gram positive

microorganisms. When glucose is present, a high intracellular level of FBP will

stimulate HPrK/P to phosphorylate the HPr at serine-46 site. This modified HPr forms

a complex with the catabolite control protein A (CcpA). The complex binds to specific

sequences called catabolite responsive elements (cre) and the transcription of a target

gene is repressed. However, under unpreferred nutritional conditions such as starvation

or grown on gluconeogenic substrate, the concentration of inorganic phosphate

increases, and phosphorylase activity of HprK/P becomes predominant. Figure adapted

from Durre (2005).

His~P

PEP pyruvate

EI EI~P

EIIA EIIA~P

EIIBEIIB~P

glucose

glucose~P

HPrHPr

metabolism

FBP

CcpA

cre gene

DNAbinding

EIIC cell membrane

ATP ADP

Ser~PHPr

HPrK/P

PTS

TRANSCRIPTION

REPRESSED

Catabolite repression

Pi

PPi


25

Until now, HPrK/P kinase activity has been observed only in one species of Clostridium

which is C. acetobutylicum, whereas, PTS activity has been reported in several species

of Clostridium (Tangney et al., 2003). A putative ptsH gene has been identified in the

genome of three species of Clostridium, C. perfringens, C. tetani, and C. beijerinckii.

Surprisingly, the ptsH gene is not followed by ptsI in the three species nor in C.

acetobutylicum which means that ptsH and ptsI are not in an operon, which is unusual

(Tangney and Mitchell, 2005). However, this situation has also been reported in some

other bacteria such as Mycoplasma capricolum and Streptomyces coelicolor (Zhu et al.,

1993). As for C. acetobutylicum, analysis of the completed genomes of C. perfringens,

C. tetani, and C. beijerinckii reveals that there is only one gene encoding for HPr and

that there is no HPr-like protein Crh found in these bacteria (Tangney and Mitchell,

2005).

Bioinformatic analysis has shown a putative C. acetobutylicum HPrK/P and this protein

has a signature sequence that is involved in the interaction with HPr and also a Walker

A box which is involved in ATP binding. The hprK gene is located downstream of an

open reading frame encoding a putative fructose 1,6-bisphosphatase (GlpX) (Tangney et

al., 2003). To date, this gene arrangement has not been observed in any other low GC

Gram positive bacterium, since as mentioned previously the hprK gene is usually linked

to the lgt gene encoding prolipoprotein diacylglyceryl transferase (Tangney et al.,

2003).

A putative gene, termed regA, encoding a CcpA-like protein in C. saccharobutylicum

was cloned into a B. subtilis ccpA mutant and was found to complement the mutation

(Davison et al., 1995). This suggested that the RegA protein is actually CcpA. Genes

encoding putative CcpA protiens have been identified in three other species of

Clostridium; C. acetobutylicum, C. tetani, and C. prefringens and all these proteins

contain an N-terminal DNA recognition binding domain (Tangney and Mitchell, 2005).

Putative cre sites have been identified in the promoter region of nearly all the pts

operons in species of Clostridium that have been studied. As an example, a cre site has

been identified in both the sucrose (scr) and lactose (lac) operons of C. acetobutylicum.

These operons are induced by sucrose and lactose respectively, and both are repressed

by glucose (Tangney and Mitchell, 2000; Yu et al., 2007). Ren et al. (2010) have

recently characterized and then disrupted the ccpA gene in C. acetobutylicum ATCC

824 in order to generate a strain that is able to utilize a mixture of sugars such as xylose

and glucose simultaneously.


26

However, the growth of the mutant strain (ccpA) was impaired with very low

consumption of glucose and xylose compared to the wild type. Also, acid

reassimilation by the mutant was inhibited which resulted in high acid accumulation.

To solve the problem of the resulting low pH, calcium carbonate was added to the

medium. As a result the mutant strain was able to grow on glucose and xylose.

Transcriptional analysis revealed that the expression level of the ctfAB operon was very

low in the ccpA mutant strain compare to the wild type (Ren et al., 2010). This

indicates that CcpA could be acting as a catabolite activator in C. acetobutylicum ATCC

824.

1.11 Fructose 1,6-bisphosphatase

Gluconeogenesis is a vital metabolic pathway that is undertaken to produce glucose

when bacteria grow on non-carbohydrate substrates such as glycerol, amino acids,

organic acids or fatty acids (Figure ‎1.5) (Brown et al., 2009). Under gluconeogenic

growth conditions, fructose 1,6-bisphosphatase (FBPase) is a key enzyme which

produces fructose 6-phosphate and orthophosphate from the hydrolysis of fructose 1,6-

bisphosphate. However, under glycolytic conditions, the reverse of the reaction is

catalyzed by 6-phosphofructokinase to produce fructose 1,6-bisphosphate from fructose

6-phosphate at the expense of ATP (Brown et al., 2009: Rittmann et al., 2003 :Jules et

al., 2009).

Five different classes of FBPase enzyme have been identified and classified based on

their primary structure. The classes I, II, and III have been reported in bacteria, class IV

in archaea, and class V in both domains of the thermophilic prokaryotes. However,

only FBPase class I enzymes have been found in eukaryotes (Brown et al., 2009: Hines

et al., 2006). These five classes of FBPases require divalent cations for activity (Mn2+

,

Zn2+

, Mg2+

) (Brown, et al., 2009; Donahue et al., 2000). The classes I, III, IV, and V

are all termed Fbpase, However, class II has been termed GlpX (Brown, et al., 2009;

Donahue et al., 2000). Most microorganisms appear to have more than one FBPase

enzyme, to date, no bacteria have the combination of class I and class III FBPases,

however, the combinations of classes I and II and classes II and III were reported

(Donahue et al., 2000; Kuznetsova et al., 2011). All the resolved crystal structures of

class I, II, IV and V have the same α-β-α-β-α secondary structure which explains why

they have the same function despite differences in primary structure (Brown, et al.,

2009; Hines et al., 2006; Nishimasu et al., 2004; Johnson et al., 2001).


27

Figure ‎1.5: Pathway of gluconeogenesis. Adapted from Berg et al., (2002).

Glucose 6-phosphatase

Phosphoglucose isomerase

Fructose 1,6-bisphosphatase (FBPase)

Aldolase

Glyceraldehyde 3-phosphate

Glyceraldehyde 3-phosphate dehyrogenase

1,3-bisphosphoglycerate

3-phosphoglycerate

Phosphoglycerate kinase

Phosphoglycerate mutase

2-phosphoglycerate

Glucose 6-phosphate

Fructose 6-phosphate

Pi

H2O

Fructose 1,6-bisphosphate (FBP)

Dihydroxyacetone phosphateTriose phosphate isomerase

Pi

H2O

Pi + NAD+

NADH

ADP

ATP

Enolase

H2O

Phosphoenolpyruvate

GTP GDP + CO2

Phosphoenolpyruvate carboxykinase

Oxaloacetate

ATP + HCO3 ADP + Pi

Pyruvate

Glycerol phosphate

NAD+

NADH + H+

Pyruvate carboxylase

Glycerol phosphate dehyrogenase

ADP

ATP

Glycerol

Glycerol kinase

Glucose


28

1.11.1 Fbpase class I

The Fbp class I has been identified in both eukaryotes and some prokaryotes. In

mammals, the FBPase enzyme is a tetramer and can be inhibited by AMP and fructose

2,6-bisphosphate. In E. coli, the class I FBPase is strongly inhibited by AMP, however,

no inhibition was detected after the addition of fructose 2,6-bisphosphate, and the

enzyme is activated at least 3-fold by PEP and sulphate. The enzyme requires Mg2+

for

activity (Brown et al., 2009; Hines et al., 2006). The FBPase protein in E. coli is the

main FBPase which is involved in gluconeogenesis and this explains why a ∆fbp strain

cannot grow on gluconeogenesis substrates such as glycerol (Donahue et al., 2000).

1.11.2 Fbpase class II (GlpX)

1.11.2.1 In E. coli

GlpX was first identified in E. coli as being encoded by one of the genes in the glpFKX

operon involved in glycerol utilization. The glpFKX operon encodes for a glycerol

facilitator (glpF), glycerol kinase (glpK), and an enzyme of unidentified function (glpX)

(Donahue et al., 2000). Brown et al, (2009) reported that another gene also encodes for

a FBPase class II enzyme (yggF). The activity of both GlpX and YggF enzymes were

strongly inhibited by inorganic phosphate and the resolved 3 dimensional structure of

the GlpX complex with phosphate confirmed that the inorganic phosphate molecule

binds to the active site of the enzyme which explains why the enzyme was strongly

inhibited. However, other effectors such as ADP, AMP and PEP each at 1 mM had no

significant effect on the activity of either enzyme (Brown et al., 2009). GlpX and YggF

proteins are homodimers of subunits of 36 and 34.3 kDa respectively. Both GlpX and

YggF enzymes show maximal activity at pH 7.5-8.0 and Mn2+

was the required divalent

cation for the activity of both enzymes. However, GlpX has higher activity and affinity

for FBP which leads to three times higher catalytic efficiency compared to YggF. In the

GlpX structure, the active site is located in the large cavity between the two protein

domains where many conserved residues are located (Brown et al., 2009).


29

1.11.2.2 In Corynebacterium glutamicum

C. glutamicum is a Gram positive, high GC content bacterium which plays an important

role in industrial amino acid production (Rittmann et al, 2003; Kalinowski et al, 2003).

This bacterium has only a class II FBPase which is a homotetramer of subunits of 35.5

kDa and shares 44% identity with E. coli GlpX (Rittmann et al., 2003). The optimal pH

to assay the activity was 7.7 and Mn2+

was required for activity. FBPase activity was

completely inhibited by 90 µM AMP, and activity was reduced to 50% after adding 360

µM PEP, 140 µM LiCl, 1.6 mM phosphate and 1.2 mM fructose 1-phosphate. This

gene was named fbp, even though the enzyme belongs to class II FBPases (Rittmann et

al., 2003). The C. glutamicum FBPase class II shows similar behaviour to the

mammalian FBPase in terms of inhibition by AMP (Hines et al., 2006; Rittmann et al.,

2003). This enzyme is an abundant protein and its synthesis does not depend on the

growth conditions. An fbp mutant could not grow on gluconeogenic substrates which

indicate that this is the major FBPase in C. glutamicum (Rittmann et al., 2003). PTS EI,

HPr and EII permeases have been identified for uptake of sugars such as fructose and

glucose/mannose, but no HPrK/P kinase was identified and also the HPr lacks the

conserved Ser-46 which indicates that the carbon catabolite repression is different in

this high GC content Gram positive bacterium from that in the low GC content Gram

positive bacteria such as B. subtilis and C. acetobutylicum (Rittmann et al., 2003).

1.11.2.3 In Mycobacterium tuberculosis

There is only one major FBPase class II encoded by the Rv1099c gene in this bacterium,

and the enzyme shares 43% identity to E. coli GlpX (Movahedzadeh et al., 2004; Gutka

et al., 2011). The optimal pH for enzyme activity is 7.3 and Mg2+

was required for

activity. AMP, citrate, Li+, PEP and ADP were tested as potential effectors. No

inhibition was seen in the presence of any of these metabolites up to 1 mM except for

Li+ which inhibited the activity by 50% at 0.2 mM, and by more than 90% at 2.5 mM

indicating that this enzyme belongs to the Li+-sensitive phosphatases (Movahedzadeh et

al., 2004; Gutka et al., 2011).


30

1.11.2.4 In Bacillus species

GlpX in B. subtilis is encoded by the ywjI gene and the enzyme shares 54% identity

with GlpX from C. glutamicum and requires Mn2+

for activity (Jules et al., 2009). The

GlpX activity was completely inhibited by addition of 1 mM PEP. A ∆ywjI strain was

able to grow on gluconeogenic substrates such as glycerol and malate. GlpX in B.

subtilis is a constitutive enzyme with expression which is not dependent on

gluconeogenic or glycolytic conditions (Jules et al., 2009). Van der Voort et al, 2008

reported that B. cereus has two glpX genes. One of them (ywjl) was affected by the

deletion of ccpA which led to relief of glucose repression and this might indicate that

ywjl is involved in gluconeogenesis. However, the second glpX was not affected by

deletion of ccpA and the presence of glucose (Van der Voort et al., 2008).

1.11.3 Fbpase class III

The first and only FBPase belonging to class III has been reported in B. subtilis (Fujita

et al., 1998). The enzyme is encoded by a monocistronic yydE gene and the expression

of this gene depends on the growth phase rather than growth conditions (Fujita et al.,

1998). Like the glpX mutant, an fbp mutant was able to grow on gluconeogenic

substrates such as glycerol indicating that each enzyme can compensate for the absence

of the other (Jules et al., 2009). The GlpX activity was completely inhibited by

additional of 1 mM PEP, however Fbp activity was increased by 30-fold after adding 1

mM PEP which indicates that GlpX and Fbp are both regulated by PEP (Jules et al.,

2009). Like glpX, fbp was expressed at a constant level independent of the growth

conditions (Jules et al., 2009; Fujita et al., 1998).

1.11.4 Fbpase class IV and V

The Fbpase class IV was first identified in Methanocaldococcus jannaschii which is

anaerobic archaeon that produces methane as a by-product (Bult et al., 1996). The

MJ0109 protein can act as both inositol monophosphatase (IMPase) and FBPase (Stec

et al., 2000). The first class V FBPase was reported in the sulphur-reducing

hyperthermophilic euryarchaeon Thermococcus kodakaraensis to be encoded by the

fbpTK gene. The enzyme showed substrate specificity for FBP only, indicating that this

is the true FBPase, unlike the bifunctional class IV IMPase/FBPase enzyme.


31

The expression of the fbpTK gene was strongly repressed under glycolytic conditions

and a fbpTK mutant was unable to grow under gluconeogenic conditions even though

impTK was constitutively expressed (Rashid et al., 2002). The activity of FbpTK was

detected without any divalent cations, but was enhanced 5-fold by the addition of 20

mM Mg2+

, and 4-fold by the addition of 1 mM Mn2+

. Also, Zn2+

increased the activity

at 1 mM but Ca2+

and Ni2+

had no effect on the activity and the optimal pH was 8.0

(Rashid et al., 2002). A recent study reported that class V FBPase is a bifunctional

fructose 1,6 bisphosphate aldolase/phosphatase (FBP A/P) enzyme (Say and Fuchs,

2010; Fushinobu et al., 2011). Fructose 1,6-bisphosphate aldolase (FBP aldolase) is an

enzyme that catalyzes FBP under glycolytic growth conditions into dihydroxyacetone

phosphate and glyceraldehyde 3-phosphate (aldo cleavage step). However, under

gluconeogenic growth conditions the same enzyme catalyzes the reverse step which is

aldol condensation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate to

produce FBP (Siebers et al., 2001; Berry and Marshall, 1993). Two different classes of

FBP aldolase have been identified and classified based on their primary structure. The

aldolase activity of FBP A/P only has irreversible step of aldol condensation of

dihydroxyacetone phosphate and glyceraldehyde 3-phosphate to produce FBP.

Bioinformatics have shown that analysis for all the archaeal genomes sequenced encode

a bifunctional FBP A/P, although the mechanism by which FBP A/P exhibits its dual

activity remains unclear (Say and Fuchs, 2010; Fushinobu et al., 2011).


32

1.12 Project aim

Interest in the putative GlpX of C. acetobutylicum started when Tangney et al, (2003)

stated that the putative hprK gene, which is a vital gene in carbon catabolite repression,

is followed by an open reading frame that might encode for a class II GlpX enzyme.

This unique gene arrangement has not been reported in any other low GC Gram positive

up to date, which might indicate that the putative GlpX enzyme plays a vital and unique

role in regulating the FBP-dependent HPrK/P kinase activity (Figure 1.4) and thus in

regulating carbon catabolite repression in C. acetobutylicum ATCC 824. For these

reasons, GlpX needs to be characterized at the biochemical and genetic levels. In

addition, the unique profile of the gene arrangement should be characterised in terms of

gene expression. Moreover, bioinformatic analysis revealed a putative class III fbp gene

in C. acetobutylicum which is an orthologue to the class III fbp gene in B. subtils.

Therefore, this gene and the encoded enzyme should be characterized and compared to

the putative class II GlpX in order to know the physiological roles of both proteins. The

overall aim of this project is to isolate by cloning, characterise and compare the

physiological function of glpX and fbp genes in the solventogenic C. acetobutylicum

ATCC 824, and also to investigate the significance of the hprK-glpX unique gene

arrangement in the bacterium.

General materials and methods

33

Chapter 2:

2 General materials and methods


34

2.1 Bacterial strains, growth conditions, primer and vector details

C. acetobutylicum ATCC 824 strain was obtained from Dr P. Soucaille of the Institut

National des Sciences Appliquées-DGBA, Toulouse, France, and was originally

obtained from American Type Culture Collection, Rockville, Maryland, USA. The E.

coli strains JB103, JB108, JLD2402, JLD2403, JLD2404, and JLD2405 were obtained

from Dr T. J. Larson of the Virginia Polytechnic Institute and State University,

Blacksburg, USA. For TOPO cloning, the E. coli strain Mach1™

-T1R

was acquired from

Invitrogen, Carlsbad, CA. For Gateway cloning the E. coli strains One Shot®

OmniMAX™ 2-T1R (Invitrogen, Carlsbad, CA) and Rosetta™

2(DE3) (Novagen

Madison, WI) were used. The E. coli strains for Ligation Independent Cloning (LIC)

were acquired from Novagen. Bacterial strains were stored at -70°C in the appropriate

glycerol media. Details of the E. coli strains used in this study are listed in Table 2.1.

Plasmids were stored at -20°C in nuclease free water (Qiagen, Valencia,

CA). Details of the plasmids used in this study are listed in Table 2.2.


35

Table ‎2.1: Details of E. coli strains used in this study and their genotypes

E. coli strain Genotype Source

TOP10F`

F´{lacIqTn10(TetR)}mcrAΔ(mrr-hsdRMS-mcrBC)

Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697

galU galK rpsL endA1 nupG

Invitrogen

Mach1™

-T1R

F-

φ80(lacZ)ΔM15 ΔlacX74 hsdR(rK

-mK

+) ΔrecA1398 endA1

tonA

Invitrogen

JB108

BL21 (DE3) zjg920::Tn10 Δfbp287

Donahue et al., 2000

JLD2402

TL524 glpX::spcr Δfbp287 zjg-920::Tn10


JLD2403

TL524 glpX::spcr fbp+ zjg-920::Tn10


JLD2404

TL524 glpX+ Δfbp287 zjg-920::Tn10


JLD2405

TL524 glpX+ fbp+ zjg-920::Tn10


JB103

F– araD139 Δ(lacZYA-argF)U169 rpsL150 deoC1 relA1

ptsF25 flbB5301 thiA1zjg-920::Tn10 Δfbp287(λ lacIq tetR+

spcR)

Lutz & Bujard, 1997

One Shot®

OmniMAX™

2-T1R

F– [proAB+ lacIq lacZΔM15 Tn10(TetR) Δ(ccdAB)] mcrA

Δ(mrr-hsdRMS-mcrBC)φ80(lacZ)ΔM15Δ(lacZYA-argF)

U169 endA1 recA1 supE44 thi-1 gyrA96 relA1 tonA panD

Invitrogen

Rosetta™

2(DE3)

F– ompT hsdSB(rB

– mB

–) gal dcm (DE3) pRARE23 (CamR)

Novagen

NovaBlue

GigaSingles™

F'[proA+

B+

lacIq

ZΔM15::Tn10 (TcR

)]endA1 hsdR17(rK12–

mK12+) supE44 thi-1 recA1 gyrA96 relA1 lac

Novagen

BL21(DE3)

pLysS

F– ompT hsdSB(rB– mB–) gal dcm (DE3) pLysS (CamR)

Novagen


36

Table ‎2.2: Plasmids used in this study

Plasmid + cloned gene Code assigned to clones after transformation

pCR2.1-TOPO + glpX pM1B, pM1W, and pM2W

pDONR™221 + glpX pMAX-glpX-1, pMAX-glpX-8, and pMAX-glpX-10

pDONR™221 + fbp pMAX-fbp-1, pMAX-fbp-2

pET-60-DEST + glpX pRosetta-glpX-6 and pRosetta-glpX-7

Pet-41 Ek/LIC + glpX pNova-glpX-3 and pNova-glpX-4

Pet-41 Ek/LIC + fbp pNova-fbp-3 and pNova-fbp-4

2.1.1 E. coli strain growth conditions

Luria-Bertani (LB) and M9 glycerol or glucose minimal medium were used to grow

these strains. The compositions of these media are shown in Table ‎2.3. Media were

autoclaved at 121°C and ampicillin or tetracycline or kanamycin was added to the

media as required after cooling to 55°C (Table 2.4). All the strains were grown at 37°C.

Table ‎2.3: Growth media composition

M9 minimal medium: 0.4 % (w/v) glycerol (per litre)

M9 salts (see below) 100 ml

Glycerol 4 g

0.1M MgSO4 10 ml

0.01M CaCl2 10 ml

dH2O to 1000 ml

M9 Minimal medium: 0.2 % (w/v) glucose (per litre)

M9 salts 100 ml

Glucose 2 g

0.1M MgSO4 10 ml

0.01M CaCl2 10 ml

dH2O to 1000 ml

M9 Salts (per litre)

Na2HPO4 60 g

KH2PO4 30 g

NaCl 5 g

NH4Cl 10 g

dH2O to 1000 ml


37

Luria-Bertani (LB) medium (per litre)

Tryptone 10 g

Yeast extract 5 g

NaCl 5 g

Table ‎2.4: List of antibiotics

Full name Abbreviation Final concentration

Ampicillin +Amp (100 µg/ml)

Tetracycline +Tet (10 µg/ml)

Kanamycin +Kan (30 µg/ml)


38

2.1.2 Primer sequences

The sequences of the forward and reverse primers used in this study to amplify specific

fragments of DNA are presented in Table 2.5. All primers were obtained from Eurofins

MWG Operon UK, except for RT-PCR control primers which were obtained from

Novagen. The extra overhang sequences for Gateway and LIC cloning primers are

underlined.

Table ‎2.5: Primer details

Designated

name

Sequence‎(5’‎– 3’)

TOPOglpX Fwd: ACTTTAGGACACTATAGCGC

Rev: TACCTGCATTTACTATCCCC

TOPOfbp Fwd: ACTTTAGGACACTATAGCGC

Rev: TACCTGCATTTACTATCCCC

GATEglpX Fwd: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTTTGATAATGATATATATCC

Rev: GGGGACCACTTTGTACAAGAAAGCTGGGTTTTCTACCACTAATTTACTTTT

GATEfbp Fwd: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCTATTAGAAAGTAAC

Rev: GGGGACCACTTTGTACAAGAAAGCTGGGTAACTTTTTACAAATTCCTT

LICfbp Fwd: GACGACGACAAGATGCTATTAGAAAGTAACACCA

Rev: GAGGAGAAGCCCGGTACTTTTTACAAATTCCTTAATAAG

LICglpX Fwd: GACGACGACAAGATGTTTGATAATGATATATCC

Rev: GAGGAGAAGCCCGGTTTCTACCACTAATTTACTTTT

RNAhprK Fwd: TATACGTCCAGGGAGGAATG

Rev: CCTTGGAAATAACCTTCGGC

RNAglpX

Fwd: TGCAGCTACAGCAATAACAG

Rev: CAAACTGTAGTCCTGGTCTG

RT-PCR

control

Fwd: TCCACCACCCTGTTGCTGTA

Rev: ACCACAGTCCATGCCATCAC

T7 promoter Fwd: TAATACGACTCACTATAGGG

-


39

2.1.3 Growth conditions for C. acetobutylicum ATCC 824

C. acetobutylicum was maintained as a spore suspension in sterile water at 4°C. Two

ml of spore suspension was transferred into a sterile test tube and heated-shocked at

80°C for 10 minutes and transferred into 20 ml of Reinforced Clostridial Medium

(RCM) which was prepared according to the manufacturer’s instructions (OXOID LTD,

England). To prepare the starter culture, the inoculated RCM was then incubated in an

anaerobic cabinet at 37°C overnight under N2:H2:CO2 (80:10:10) mixed gas atmosphere.

Five ml of starter culture was then transferred into 100 ml of Clostridial Basal Medium

(CBM) [casein hydrolysate, 4 g; MgSO4.7H2O, 0.2 g; FeSO4.7H2O, 10 mg;

MnSO4.4H2O, 10 mg; α-aminobenzoic acid, 1 mg; thiamine-HCl, 1 mg; d-biotin, 2 µg;

KH2PO4, 0.5 g; 5 mM of glucose in 1 L of dH2O] and incubated at 37°C under the same

anaerobic conditions. The cells were harvested by transferring them into 1.5 ml

microcentrifuge tube, centrifuged at 18000 xg in room temperature for 10 minutes and

the supernatant was discarded and the cell pellet was kept at -70°C.

2.2 Chromosomal DNA extraction from C. acetobutylicum ATCC 824

The chromosomal DNA was extracted by using a DNA Isolation Kit for Gram-positive

bacteria (Gentra®, Cat# D-6000A) with some modifications in the protocol. Cell

Suspension Solution (300 µl) was added to the C. acetobutylicum cell pellet that was

prepared as described above, and then the cell pellet was pipetted up and down until the

cells were suspended. Lytic Enzyme Solution (1.5 µl) was added to the mix and

vortexed for 30 seconds, and then incubated on the shaking incubator at 100 rpm for 30

minutes at 37°C. The reaction mix was centrifuged again at 18000 xg for 1 minute and

the supernatant was discarded.

Cell Lysis Solution (300 µl) was added to the cell pellet, to break open the cells and

release the DNA, and then gently pipetted up and down to lyse the cells, 1.5 µl of

RNase A Solution was added to the lysed cells, and incubated in a shaking incubator at

100 rpm for 1 hour at 37°C. Protein Precipitation Solution (100 µl) was added to the

reaction mix and vortexed for 30 seconds and centrifuged at 18000 xg for 3 minutes, a

white pellet containing the precipitated proteins was then formed. The supernatant

containing the DNA was transferred into a sterile 1.5 ml microcentrifuge tube

containing 300 µl of 100% isopropanol, vortexed for 30 seconds, and then centrifuged

at 18000 xg for 1 minute.


40

The supernatant was discarded and the pellet which contained the genomic DNA was

resuspended again in 300 µl of 70% (v/v) ethanol, vortexed for 30 seconds, and then

centrifuged at 18000 xg for 1 minute. The supernatant was discarded and the pellet was

hydrated in 60 µl of DNA Hydration solution, then incubated for 1 hour at 65°C and the

purified genomic DNA was stored at -20°C.

2.3 Polymerase Chain Reaction (PCR) for TOPO™ TA cloning

C. acetobutylicum ATCC 824 chromosomal DNA was purified to be used as DNA

template in the PCR reaction. The total volume of the PCR reaction was 50 µl which

consisted of; 1 µl of chromosomal DNA, 1 µl of each of the TOPO forward and reverse

primers for both glpX and fbp genes, 10 µl of 5 × PCR buffer [100 Mm of dNTPs, 20

µl; 10 × Buffer (Stratagene®, Cat# 600400), 230 µl; dH2O, 250 µl] and 36.5 µl of

deionised water. After the first denaturation stage of 95°C for 5 minutes, 0.5 µl of

Easy-A high fidelity PCR cloning enzyme (Stratagene®, Cat# 600400) was added to the

PCR mixture. The second stage was 30 cycles of 1 minute at 95°C, 1 minute at the

appropriate annealing temperature and 3 minutes at 72°C. The final stage was 10

minutes at 72°C to complete primer extension.

2.4 Gel electrophoresis

To enable visualisation of nucleic acids, 5 µl of PCR product were mixed with 2 µl of

DNA loading buffer (Bioline, Cat# BIO-37045). Then 7 µl of the resultant mixture was

loaded onto a 1% (w/v) agarose gel (containing 1 µl of Ethidium Bromide at 10 mg/ml)

along with Hyperladder I (Bioline), as size standards and run at 60 V for 1 hour, and the

DNA in the gels were visualised by UV light.

2.5 TOPO™ cloning procedure

A TOPO TA Cloning®

Kit (Invitrogen, Cat# K4500-01) was used to perform the

cloning of both glpX and fbp genes. Taq polymerase has a non-template dependent

terminal transferase activity that adds a single deoxyadenosine (A) to the 3’ ends of the

PCR product. The pCR2.1-TOPO vector possesses single overhanging 3’

deoxythymidine (T) residues. This enables cloning of the PCR product into the

pCR2.1-TOPO vector (appendix 7.4).


41

Fresh PCR products (see section 2.3) (3 µl) were mixed with 1 µl water, 1 µl Salt

Solution and 1 µl pCR2.1-TOPO vector (all provided from the TOPO kit). The TOPO

cloning reaction was incubated for 15 minutes at room temperature with the protocol

carried out as per manufacturer’s instructions.

2.6 Transformation of chemically competent E. coli

A vial of Mach1™

-T1R competent

cells (Table ‎2.1) stored at -70°C was thawed on ice

prior to use. After thawing, 2 µl of TOPO cloning reaction was added into the vial and

incubated for 10 minutes on ice and then the cells were heat-shocked for 30 seconds at

42°C. Immediately the vial was placed on ice for 2 minutes and then a 250 µl of S.O.C

medium (provided from the TOPO kit) was added at room temperature. The vial was

incubated horizontally at 37°C on a shaking incubator at 170 rpm for 1 hour. LB plates

(Table ‎2.3) supplemented with 40 µl/plate of 40 µg/ml X-gal and (+Amp) were

incubated for 1 hour at 37°C to pre-warm the plates. Aliquots from the transformation

mixture (50 µl or 100 µl) were then spread onto the surface of the agar. The plates were

incubated overnight at 37°C.

2.7 Screening of clones

2.7.1 PCR analysis

White colonies contain an inserted gene which disrupts the lacZ gene fragment on the

pCR2.1-TOPO vector. Therefore the white colonies were tested for harbouring a

plasmid containing the desired PCR insert. This was done by transferring a colony,

using a sterile toothpick, to 20 µl of dH2O in a 1.5 ml microcentrifuge tube and heating

to 100°C in a heat block for 10 minutes. The mixture was transferred to ice for 10

minutes to cool down and then centrifuged at 18000 xg for 2 minutes. After the

centrifugation, the supernatant was used as the DNA template for a PCR reaction. The

PCR screening was carried out using the same primers that were used in the PCR

amplification with 1 µl of the supernatant. The reaction mix and conditions were the

same as previously described in Section 2.3. For determination of the orientation of the

insert, the T7 promoter primer (Table 2.5) was used in conjunction with one of the

TOPOglpX primers. PCR products were visualised following electrophoresis in 1%

(w/v) agarose gels in 1 × Tris-acetate-EDTA (TAE) buffer at 60 V.


42

2.7.2 Plasmid purification

Plasmids were purified using the Qiagen Plasmid Midi Kit (Cat# 12145) as described in

the manufacturer’s protocol. A single colony of the cells containing the plasmid of

interest was transferred to 10 ml of LB Broth supplemented with appropriate antibiotic

and incubated overnight with shaking at 37°C. Cell suspension (1 ml) was transferred

into 100 ml of LB Broth supplemented with appropriate antibiotic and incubated again

overnight with shaking at 37°C. The cell suspension (50 ml) was then pelleted by

centrifuging at 2700 x g for 10 minutes at 4°C. The pellet was resuspended in 4 ml of

Buffer P1 and vigorously shaken for 1 minute. Lysis Buffer (4 ml, Buffer P2) was

added to the resuspended pellet and mixed by inverting 6 times and incubated at room

temperature for 5 minutes. Then, 4 ml of chilled Buffer P3 was added to the mixture to

stop the lysis reaction and incubated on ice for 15 minutes. The reaction mixture was

centrifuged at 20000 xg for 30 minutes at 4°C, the supernatant was transferred to

another tube and centrifuged at 2700 xg for 10 minutes at 4°C and the supernatant was

kept and the pellet discarded. The purification column (QIAGEN-tip 100) was

equilibrated by adding 4 ml of Buffer QBT to the purification column and allowing it to

empty by gravity flow.

The supernatant was applied to the purification column and allowed to move through

the column by gravity flow and then the column was washed twice by applying 10 ml of

Buffer QC. The plasmid was eluted by adding 5 ml of Buffer QF and collected in a 15

ml tube. Isopropanol (3.5 ml) was added and centrifuged at 2700 xg for 1 hour at 4°C

to precipitate the plasmid. The plasmid pellet was washed by the addition of 2 ml of

70% (v/v) ethanol and centrifuged at 2700 xg for 30 minutes, after which the

supernatant was discarded. The pellet was resuspended in 50 µl of deionized water. The

concetraction of plasmid DNA was quantified by comparing it to concentration of

Hyperladder I (Bioline).

2.7.3 DNA Sequencing

All DNA sequencing was conducted commercially by Beckman Coulter Genomics.

Plasmid DNA was sent in 20 µl of nuclease free dH2O at a concentration of 50 ng/µl.


43

2.7.4 Restriction analysis

Colonies thought to possess a PCR insert, were grown in 100 ml LB Broth (+Amp).

The Qiagen Plasmid Midi Kit was used to purify the recombinant plasmid. After

purification, 5 µl of the sample was electrophoresed in a 1% (w/v) agarose gel in 1×

TAE buffer at 60 V for 1 hour, to check whether the plasmid of interest was purified or

not. Separate digestion reactions using EcoRI (Fermentas, Cat# ER0271, cutting sites:

5'...G↓A A T T C...3' and 3'...C T T A A↓G...5') and HindIII (Fermentas, Cat# ER0501,

cutting sites: 5'...A↓A G C T T...3' and 3'...T T C G A↓A...5') restriction enzymes were

performed to determine the orientation of the insert. The reaction mixture (Table 2.6)

was incubated at 37°C overnight before being visualised by electrophoresis. The

restriction digest sites of HindIII and EcoRI in the vector are shown in (Appendix 7.4).

Table ‎2.6: Composition of the restriction digest mixture

Substance Volume (µl)

10 × buffer of EcoRI or HindIII 2

Plasmid DNA 5

Restriction digest enzyme 1

dH2O 12

Total volume 20

2.7.5 Transformation of E. coli mutant strains

The E. coli strains JB103, JB108, JLD2402, JLD2403, JLD2404, and JLD2405 were

grown on Nutrient Agar. Single colonies of these strains were transferred to 10 ml of

LB supplemented with tetracycline (+Tet), and incubated on the shaking incubator at

170 rpm overnight at 37°C. After that, 1 ml of overnight culture was transferred into

100 ml of LB (+Tet) and incubated at 37°C (170 rpm) for 4 hours until the OD600

reached 0.6. The culture (50 ml) was transferred into a 50 ml polypropylene tube and

then incubated on ice for 10 minutes. The cells were centrifuged at 1700 xg at 4°C for

10 minutes. The supernatant was discarded and the pellet was resuspended in 10 ml of

ice-cold 0.1 M CaCl2 and again incubated on ice for 10 minutes. The suspension was

centrifuged again at 1700 xg at 4°C for 10 minutes and then the cells were resuspended

in 2 ml of the same ice-cold 0.1M CaCl2.


44

Purified recombinant of interest plasmid (2.5-5 µl) was added to 200 µl of cell

suspension using a chilled pipette tip and then mixed gently and incubated on ice for 10

minutes. After that, the reaction mixture was heat shocked for 2 minutes at 42°C in a

water bath and placed on ice again for 5 minutes. An 800 µl aliquot of LB broth was

added to the mixture and incubated on the shaking incubator (100 rpm) at 37°C for 1

hour. After the incubation, 100 µl of the mixture were spread onto LB plates (+Amp

and +Tet) and incubated again at 37°C overnight. The colonies which formed were

given a reference number and transferred onto M9 glucose (+Amp and +Tet), LB

(+Amp and +Tet) and M9 glycerol (+Amp and +Tet) plates, incubated overnight at

37°C and then PCR screened the same way as described in section 2.7.1.


45

2.7.6 Fructose 1,6-bisphosphatase assay

A colony that contains a gene of interest was transferred using a sterile toothpick into 10

ml LB broth (+Kan) and incubated overnight with shaking at 200 rpm at 37°C. The

overnight culture (1 ml) was transferred into 500 ml of Overnight ExpressTM

Instant LB

medium and incubated overnight with shaking at 200 rpm at 37°C [ glycerol, 10 g;

Overnight ExpressTM

Instant LB medium, 45 g in 1 L of dH2O] (Novagen, Cat#

71757). The 500 ml culture was centrifuged at 1700 xg for 10 minutes at 4°C. The

supernatant was discarded and the cell pellet was suspended in ice-cold 20 mM Tris-Cl

(8 pH) and centrifuged again similar to the previous conditions. The suspension was

transferred into a 50 ml polypropylene tube and centrifuged at 1700 xg at 4°C for 10

minutes. The weight of the cell pellet was recorded before it was stored at -20°C. The

pellet was thawed on ice and resuspended in 50 mM Tris-Cl (pH8) containing 1 mM

dithiothreitol (DTT) and 5 mM MgCl2 to reach a concentration of 3 ml/g pellet. The

cells were broken twice using a French Pressure Cell (SLM instruments, INC.) then

centrifuged at 2700 xg for 10 minutes at 4°C. Finally, the supernatant was transferred

into 15 ml polypropylene tube and stored at -20°C. The cell extracts were assayed

spectrophotometrically, by using a Beckman Coulter DU 800 spectrophotometer at 340

nm at 37°C, for FBPase activity which involved measuring the production of fructose 6-

phosphate coupled to the reduction of NADP+ in the presence of glucose 6-phosphate

dehydrogenase (G6PDH) (Sigma, 4.6 mg protein/ml; 715 units/mg protein, Cat#

G8404-1KU) and phosphoglucose isomerase (PGI) (Sigma, 10.1 mg protein/ml; 547

units/ mg protein, Cat# P5381-1KU) as follows:

The formation of NADPH in the final step of the reaction was measured by the increase

of the absorbance at 340 nm and FBPase specific activity is calculated using the molar

extinction coefficient of NADPH which is 6.22 X 10-3

nmoles (Jules et al., 2009;

Fraenkel et al., 1966; Babul et al., 1983) as follows:


46

The activity of the coupling enzymes glucose 6-phosphate dehydrogenase and

phosphoglucose isomerase were assayed in same way but instead of the FBP, 20 µl of

F-6-P (500 mM) was added to the reaction mixture to check whether the enzymes were

able to form NADPH. The composition of the assay reaction mixture is shown in Table

2.7.

Table ‎2.7: The composition of the fructose bisphosphatase assay reaction mixture

Substance Volume (µl)

1 M Tris-Cl pH8 50

1 M MgCl2 10

200 mM DTT 5

0.1 mM EDTA 4

50 mM Fbp 4

50 mM NADP 4

Coupling enzymes 1 of each

Cell extract 50

dH2O 871

Total reaction volume 1000

2.7.6.1 Microbuiret protien assay

A microbuiret assay was used to measure the concentration of the protein present in the

crude extract. A serial standard was made using BSA as shown in Table 2.8, in 1 ml

tube with the addition of 240 µl of 40% (w/v) NaOH. After that, 680 µl of water and 63

µl of 1% (w/v) CuSO4 .5 H2O were added to the mixture and then these were incubated

in the 37°C water bath for 15 minutes. The standard samples were read at 310 and 390

nm to calculate the concentration of the standards for creating a calibration curve. The

concentration of protein in 10 µl of each crude extract sample was measured in same

way but instead of BSA, which was used as standard, crude extract was used. The

protein concentration of crude extract was calculated by interpolation from the

standards calibration curve (appendix 7.9).


47

Table ‎2.8: Different amounts of BSA and dH2O that were needed in the 1 ml assay

mixture.

BSA (10 mg/ml) µl dH2O µl

20 0

15 5

10 10

5 15

0 20


48

2.8 Cloning of the glpX and fbp genes in the Gateway expression system

The Gateway expression system (Invitrogen, Cat# 12535-029) is based on two cycles of

bacteriophage lambda in E. coli. The bacteriophage lambda can either enter, a lytic

cycle which results in viral replication and destruction of the infected cell, or a

lysogenic cycle in which it integrates itself into the host genomic DNA at a specific site.

Also in the lysogenic cycle, the phage genome can either be transmitted into daughter

cells or it can be induced to go back again to the lytic cycle from which the phage

genome can be excised from the host genomic DNA. This system consists of two

reactions. The first reaction is BP reaction, which is the integration process (lysogenic

pathway) that facilitates recombination of attB-flanked PCR product with pDONR™

vector to produce an attL-containing entry clone, and this reaction or pathway is

catalyzed by bacteriophage lambda Integrase (Int) and E. coli Integration Host Factor

(IHF) proteins (BP Clonase™

II enzyme mix). The second reaction is the LR reaction

which is the excision process (lytic pathway) that facilitates recombination of an entry

clone with a destination vector, and this reaction or pathway is catalyzed by

bacteriophage lambda Excisionase (Xis), Integrase (Int) and E. coli Integration Host

Factor (IHF) proteins.

2.8.1 BP cloning reaction

Primers were designed to amplify C. acetobutylicum glpX and fbp genes flanked by

attB1 and attB2 overhangs (Table ‎2.5), that can recombine with the donor vector

(pDONR™

221) at attP1 and attP2 sites to produce entry clones with two sites, attL1 and

attL2. Both glpX and fbp genes were amplified to produce attB-PCR products for the

BP reaction as previously described in section 2.3. The attB-PCR product (1 µl) was

mixed with 1 µl of the donor vector (pDONR™221) and 6 μl Tris-EDTA (TE) buffer

(pH 8.0) was added to the mixture, both provided with the kit. The reaction was

performed in a 1.5 ml microcentrifuge tube in room temperature. The BP Clonase™

II

enzyme mix was removed from the freezer and left on ice 2 minutes for thawing, and

then mixed by vortexing twice for 2 seconds, 2 µl of BP Clonase™ II enzyme mix was

added to the reaction mixture and mixed by vortexing twice for 2 seconds. The reaction

mixture was incubated for 1 hour at room temperature, and after that 1 µl of proteinase

K solution was added and incubated again for 10 minutes at 37°C.


49

2.8.2 Transformation of chemically competent cells

One vial of One Shot® OmniMAX

™ 2-T1R Chemically Competent E. coli (Invitrogen,

Cat# 12535-029) was left on ice to thaw for 5 minutes and then 1 µl of BP reaction

mixture was added to the One Shot® OmniMAX

™ 2-T1R cells and mixed gently. The

vial was incubated on ice for 30 minutes and then heat shocked in a water bath for 30

seconds at 42°C, and placed again on ice for 2 minutes. S.O.C medium (250 µl) was

added to the vial at room temperature, and incubated for 1 hour at 37°C, shaking

horizontally at 200 rpm. After that, 20 µl of the transformation mixture was spread onto

LB plates (+Kan) and left in the incubator overnight at 37°C.

2.8.3 LR reaction

Plasmids of fbp and glpX were purified as described in section 2.7.2 using the Qiagen

plasmid purification midi kit, and sent for sequencing to Eurofins MWG Operon. The

following reaction was performed in a 1.5 ml microcentrifuge tube at room temperature,

1 µl of 150 ng purified plasmid (entry clone) was mixed with 1 µl of pET-60-DEST

vector, and then 6 µl of TE buffer, pH 8.0 was added to the mixture. The LR Clonase™

II enzyme mix was removed from the freezer and left on ice for 2 minutes for thawing,

and then mixed by vortexing twice for 2 seconds, 2 µl of LR Clonase™

II enzyme mix

was added to the reaction mixture and mixed by vortexing twice for 2 seconds. The

reaction mixture was incubated for 1 hour at room temperature, after that 1 µl of

proteinase K solution was added and then incubated again for 10 minutes at 37°C.

2.8.4 Transformation into expression host

One vial of Rosetta™

2(DE3) Competent Cells (Novagen, Cat# 71397) was left on ice to

thaw for 5 minutes and then 1 µl of LR reaction mixture was added to the Rosetta™

2(DE3) Competent Cells and mixed gently. The transformation was performed as

described in section 2.8.2.


50

2.9 Cloning of glpX and fbp by Ligation-Independent Cloning (LIC)

Novagen ligation-independent cloning (LIC) allows directional cloning of the insert

with no need for ligation or restriction digest reaction. This system uses the activity of

the bacteriophage T4 DNA polymerase enzyme to make specific 15-base single

stranded overhangs in the Ek/LIC vector. Complementary overhangs in the insert must

be present by adding suitable 5’ extension into the primers (Table 2.5). To create

specific vector compatible overhangs, purified insert DNA must be treated with T4

DNA polymerase in the presence dATP. Transformation into competent E. coli was

carried out after cloning.

2.9.1 LIC cloning reaction

Primers LICglpX and LICfbp were designed for amplification of glpX and fbp genes

flanked by specific 13-bases at the ends which were complementary to the overhangs in

the Ek/LIC vector (Table ‎2.5). Hot Start DNA Polymerase (KOD) (Novagen, Cat#

71086-5) was used for amplification of both glpX and fbp genes from the purified C.

acetobutylicum chromosomal DNA. For reaction set up and cycle conditions see Table

2.9 and Table 2.10 respectively.

Table ‎2.9: Hot start DNA polymerase reaction setup.

Component Volume‎(μl)

10X Buffer for KOD Hot Start DNA Polymerase 5

25 mM MgSO4 3

dNTPs (2 mM each) 5

PCR Grade Water 33

10 pmol/μl Sense Primer 1

10 pmol/μl Anti-Sense Primer 1

Template DNA 1

KOD Hot Start DNA Polymerase (1 U/μl) 1



51

Table ‎2.10: Temperatures and times that were used for amplification by KOD Hot

Start DNA Polymerase.

Cycle Temperature (°C) Time

1-Polymerase activation 95 2 min

2-Denature 95 20 s

3-Annealing

51 (fbp)

56 (glpX)

10 s (fbp)

10 s (glpX)

4-Extension 70 (glpX)

70 (fbp)

20 s (glpX)

40 s (fbp)

Repeat steps 2–4 30 times

5-Final extension 70 3 min

2.9.2 Purification and precipitation of PCR product

After amplification, PCR products were purified to remove any contaminating DNA.

Therefore, DNA extraction from agarose gel with Ultrafree®

-DA kit (Millipore, Cat#

42600) was used for purification of the PCR product. PCR products (60 µl) were run in

a modified TAE electrophoresis gel [40 mM Tris-acetate, pH 8.0, 0.1 mM Na2EDTA]

for 1 hour alongside hyperladder I, and then the gel was placed on a UV box to locate

the bands. The bands were cut out using a blade and transferred into a Gel Nebulizer

and the device was sealed with the cap attached to the vial, centrifuged at 6800 xg for

10 minutes, and finally the supernatant which contained the DNA was captured in the

vial and the Gel Nebulizer which contained the agarose gel was discarded.

To inactivate the polymerase enzyme and remove dNTPs, isopropanol precipitation was

used, 80 µl of gel extracted DNA was added to 80 µl of phenol-chloroform isoamyl

alcohol, 24:1), mixed by vortexing for 1 minute, and centrifuged at 18000 xg for 1

minute and then the supernatant mixed with an equal volume of chloroform to remove

any residue of phenol, and centrifuged at 18000 xg for 5 minutes. The top phase of the

supernatant which contained the DNA was transferred into a new 1.5 ml tube, and 0.1

volume of 3 M sodium acetate pH 5.2 and 1 volume isopropanol were added, mixed by

vortexing and incubated at room temperature for 5 minutes to precipitate DNA.


52

After centrifugation at 18000 xg for 5 minutes, the supernatant was removed and the

DNA pellet was washed with 70% (v/v) ethanol, spin at 18000 xg for 5 minutes, and

then the DNA pellet was resuspended in 30 µl TE buffer [10 mM Tris-HCl, 0.1 mM

EDTA pH 8.0] which was provided with the kit.

2.9.3 T4 DNA Polymerase treatment of the purified PCR product

To generate compatible overhangs on the purified PCR product, T4 DNA Polymerase

(Novagen, Cat# 71086-5) was added to the reaction mix as shown in Table 2.11. To

start the reaction, the enzymes was added to the reaction mix, stirred with a pipette tip

and incubated at room temperture for 30 minutes. The enzyme was inactivated by

incubating at 75°C for 20 minutes.

Table ‎2.11: T4 DNA polymerase reaction setup.


100 ng/µl Purified PCR product 4

10X T4 DNA Polymerase Buffer 2

25 mM dATP 2

100 mM DTT 1

Nuclease-free Water 10.6

2.5 U/ml T4 DNA Polymerase 0.4


The treated PCR product (1 µl) was added to the pET-41 Ek/LIC vector (2 µl) in order

to perform the annealing reaction. The reaction mix was incubated at room temperature

for 5 minutes and finally, 1 µl of 25 mM EDTA was added to stop the reaction and

preserve recombinant vector.

2.9.4 Transformation into the cloning strain

The vector that contained the cloned PCR product was transformed into NovaBlue

GigaSingles™

Competent Cells which harbor recA and endA mutations to increase the

transformation efficiency and yield of recombinant plasmid DNA (Novagen, Cat#

71086-5) as described in section 2.8.2. The recombinant vector was purified as

described in section 2.7.2.


53

2.9.5 Transformation into the expression strain

The BL21 (DE3) pLysS Competent Cells (Novagen, Cat# 69451) were used to

overexpress GlpX and Fbp proteins. The transformation was carried out as described in

the instructions of the competent cell kit. The tube that contained the competent cells

was taken from the -70°C freezer and immediately placed on ice for 5 minutes to thaw.

Then 1 µl of purified plasmid (10 ng/μl) was added to the cells, mixed gently and placed

on ice. The tube was incubated on ice for 5 minutes, and the heated in a water bath at

42°C for 30 seconds, returned to ice for 2 minutes and then 250 µl of S.O.C medium

was added. The tube was incubated at 37°C for 1 hour at 200 rpm in the shaking

incubator, and then 100 μl of the mixture was plated on a selective medium which

contained (+Kan), and incubated overnight at 37°C.

2.9.6 PCR Screening

PCR screening was conducted as per Section 2.7.1 to verify whether the plasmid was

transferred to the BL21 (DE3) pLysS Competent Cells.

2.9.7 Expression of the Fbp and GlpX proteins

After the fbp and glpX genes were identified as being cloned correctly in the Pet-41

Ek/LIC plasmid, the Fbp and GlpX proteins were expressed as His tagged proteins

using Overnight ExpressTM

Instant LB media (Novagen, Cat# 71757). The procedure

for the Fbp and GlpX proteins expression are summarized as follows.

A colony of recombinant BL21 cells containing either fbp or glpX gene was transferred

using a sterile toothpick into 10 ml LB Broth (+Kan) and was incubated overnight with

shaking at 200 rpm at 37°C. The overnight culture (1 ml) was transferred into 500 ml

of Overnight ExpressTM

Instant LB media, and incubated overnight with shaking at 200

rpm at 37°C. After induction, cells were centrifuged at 20000 xg at 4°C for 10 minutes.

The supernatants were discarded and the pellets were washed by resuspending in

distilled water, and a second centrifugation was carried out at 2700 xg at 4°C for 20

minutes and finally the supernatants were discarded and the cell pellets were

resuspended in Ni-Nitrilotriacetic acid (NTA) His•Bind Matrix buffer (3 ml for each 1 g

of cell pellet) [50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 10 mM imidazole].


54

The suspended cells were lysed using a French Pressure Cell Press® under 1000 PSI

twice and then the lysates were centrifuged at 2700 xg at 4°C for 20 minutes to separate

the soluble fraction from insoluble material.

2.9.8 Purification of protien under native conditions using immobilizing metal-

affinity chromatography (IMAC)

This purification system is based on a separation technique which uses chelating

compounds with immobilized metal ions that covalently bind to amino acid residues

exposed on the surface of the target protein (Gaberc-Porekar and Menart, 2001). This

purification system uses nitrilotriacetic acid (NTA) as the chelating compound, Ni2+

as

the immobilized metal ion and the hexahistidine tags as amino acid residues exposed on

the surface of the target protein (Figure ‎2.1).

Figure ‎2.1: Schematic‎ illustration‎of‎a‎protein‎with‎a‎His•Tag‎attached‎at‎ the‎N-

terminal binding to a metal-chelated affinity support (NTA-Ni).

Strong binding of a protein onto the IMAC matrix is achieved predominantly by multi-

point attachment of the engineered surface histidine tag which is located at the N- or

and C-terminus of the protein. Figure adapted from Gaberc-Porekar and Menart,

(2001).

The His tagged Fbp and GlpX proteins were purified using BugBuster® Ni-NTA

His•Bind Purification kit (Novagen, Cat# 70751-3) under native conditions by

following the manufacturer’s protocol with the modification of using 1 M imidazole in

the third and fourth elutions and being left for 1 hour to collect the elution.


55

The 50% Ni-NTA His•Bind slurry that was provided with the kit (1 ml) was added to 4

ml of 1X Ni-NTA Bind Buffer and mixed gently and left for 20 minutes to settle. A 4

ml of soluble lysate was added to the Ni-NTA His•Bind slurry and mixed gently and

incubated 1 hour at 4°C.

After incubation, the mixture was loaded into a column with the outlet capped and left

for 20 minutes at room temperature and then the cap was removed and the flow-through

was collected in a 15 ml tube. The washing step was carried out twice by added 4 ml of

1X Ni-NTA Wash Buffer [50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 20 mM imidazole]

and the washing fractions were collected. Finally, 4 × 0.5 ml of Ni-NTA Elution Buffer

[50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 250 mM imidazole] was added to elute the

His tagged protein and collected in four tubes.

2.9.9 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

A 10 μl of both purified proteins were added to 10 μl of 2 × SDS protein samples

loading buffer and heated in a heating block at 100°C for 10 minutes, then the mixtures

were loaded on the gel. Also 10 μl of insoluble fractions of the cell extract were

removed and added to the separate volumes of 2 × SDS protein samples loading buffer

and heated in a heating block at 100°C for 10 minutes, then the mixtures were also

loaded on the gel. The AE-6450 Dual Mini Slab Kit (ATTO, Cat# 2322222) cassette

was assembled for mini gel as described in the manufacturer`s manual and the SDS-

PAGE gel was prepared with the stacking gel at the top and the running gel at the

bottom. A 7% and 4% (v/v) of acrylamide/bis-acrylamide (30%, Sigma) in buffer were

used in making the stacking and running gel, respectively. First, the running gel was

prepared by loading around 4/5 of the cassette and then a layer of 98% (v/v) butanol

was added above the running gel and left for 1 hour. After the running gel had

polymerized, the butanol was discarded and replaced by the stacking gel and left for 45

minutes to polymerise.

The 7% (v/v) running gel contained the following (30 ml): 15.3 ml of H2O, 7.5 ml of

1.5 M Tirs-HCl (pH 6.8), 140 µl of 20% (w/v) SDS, 6.9 ml of acrylamide/bis-

acrylamide (30%, Sigma), 140 µl 10% (w/v) ammonium persulfate, 20 µl of

Tetramethylethylenediamine (TEMED).


56

The 4% (v/v) stacking gel contained the following (5.05 ml): 3.075 ml of H2O, 1.25 ml

of 1.5 M Tirs-HCl (pH 6.8), 25 µl of 20% (w/v) SDS, 670 µl of acrylamide/ bis-

acrylamide (30 %, Sigma), 25 µl 10% (w/v) ammonium persulfate and 5 µl of TEMED.

After the 20 µl samples were loaded alongside 10 µl of HyperPage Prestained Protein

Marker (Cat# 33066, Bioline), the gel was run in the SDS-PAGE tank containing a

reservoir buffer [30.3 g Tris base; 144 g Glycine; 10 g SDS with 1L dH2O] at 200 V

and left for 2 hours. The gel was then removed from the cassette and placed in a box

containing Coomassie blue staining buffer [45% (v/v) methanol; 10% (v/v) acetic acid;

0.2% (w/v) Comassie Brilliant Blue; 44.8% (v/v) water] to stain the gel and left for 1

hour at 37°C, followed by overnight destaining at room temperature in the destaining

buffer [60% (v/v) methanol; 10% (v/v) acetic acid; 30% (v/v) water].

2.9.10 Dialyzing of the recombinant proteins

The recombinant proteins were dialyzed by using Dialysis Cassette Slide-A-Lyzers (3

ml) (Fisher Scientific, Cat# 66382). Dialysis cassette is a single use, self-floating

syringe-loadable unit. The dialysis cassette membrane is made from regenerated

cellulose that allows a complete retention of proteins and macromolecules that are larger

than 10 kDa, at the same time, allowing the removal of unwanted buffer. These dialysis

cassettes can be loaded with up to 3 ml of the sample needs to be dialyzed.

The recombinant proteins were dialyzed as described in the manufacturer`s protocol. A

5 ml syringe was filled with the recombinant protein up to 3 ml and injected through the

gasket of a needle port at the cassette’s corner by the needle. After that, the needle was

removed and the gasket resealed and the cassette corner was marked to note which

needle port had been used. The cassette was placed vertically with foam attached in 1

litre of dialysis buffer and stirred gently, and was left to dialyze for overnight at 4°C.

The dialysis cassette was removed from the dialysis buffer, and then the needle was

inserted into an unused needle port to remove the dialyzed recombinant protein.


57

2.9.11 Cleaving the fusion protein with recombinant enterokinase

The Enterokinase Cleavage Capture Kit from Novagen (Cat# 69067) can be used in

order to remove tags from proteins in the N-terminal (GST.Tag, His.Tag and S.Tag).

Recombinant enterokinase (rEK) is a catalytic subunit of bovine enterokinase which

cleaves after the C-terminal end of a lysine residue in the sequence Asp-Asp-Asp-Asp-

Lys↓-insert. After the cleavage of the target protein, rEK is removed from the reaction

by using affinity capture on EKapture™

Agarose and finally the agarose is removed

using a spin-filtration tube. First, small scale optimization is needed to estimate the

appropriate amount of target protein Table 2.12.

Table ‎2.12: Reaction components for small scale optimization incubated in room

or 37°C temperature.


10X rEK Cleavage/Capture Buffer 5

Target protein 20, 10

Diluted rEK (1 unit/μl) 1, 2

Deionized water Until the total volume reached 50

2.10 Growth of C. acetobutylicum ATCC 842 for RNA extraction

C. acetobutylicum ATCC 842 was kept as spores in the cold room 4°C in sterile water.

Spore suspension (2 ml) was transferred into an empty sterile tube and heat shocked at

80°C for 10 minutes, and then transferred into 20 ml of Reinforced Clostridial Medium,

(RCM, Oxoid) and incubated at 37°C for 2 days in the anaerobic cabinet (Forma

Scientific, Marietta, Ohio) to prepare starter cultures. Starter culture (3 ml) was

transferred into 100 ml of Clostridial Basal Medium (CBM) and incubated at 37°C in

the anaerobic cabinet overnight. Overnight culture (1.5 ml) was transferred into a

microcentrifuge tube, 1 ml of RNA Protect Bacteria Reagent (Qiagen, Cat# 76506) was

added and incubated at room temperature for 10 minutes, and then centrifuged for 10

minutes at 10600 x g. The supernatant was discarded and the pellet was immediately

frozen in liquid nitrogen and then stored at -70°C.


58

2.10.1 RNA extraction for C. acetobutylicum ATCC 824

The RNA was extracted by using RNeasy Mini Kit as described in the manufacturer’s

protocol (Qiagen, Cat# 76506). A tube of cell pellet was transferred from the -70°C

freezer into ice to thaw for 5 minutes, and then 200 µl of QIAGEN Proteinase K was

added and the pellet was resuspended by pipetting up and down several times. The

mixture was mixed by vortexing for 10 seconds and then incubated at room temperature

for 10 minutes, during this period of incubation, the sample was vortexed for 10

seconds every 2 minutes. The RLT Buffer (700 µl) was added to the mixture and mixed

by vortexing then, 500 µl of ethanol (96%) was added to the sample and mixed by

pipetting. Lysate (700 µl) was transferred into an RNeasy Mini spin column and placed

into a 2 ml collection tube, and centrifuged for 15 seconds at 10600 xg, and the flow-

through was discarded. The Buffer RW1 (700 µl) was added to the RNeasy Mini spin

column, and centrifuged for 15 seconds at 10600 xg to wash the membrane of the spin

column and the flow-through and the collection tube were discarded. The RNeasy

Mini spin column was placed in the top of a new 2 ml collection tube and 500 µl of

Buffer RPE was added and centrifuged for 15 seconds at 10600 xg to wash the spin

column, and again the flow-through was discarded. Again 500 µl of Buffer RPE was

added to the RNeasy Mini spin column and centrifuged for 15 seconds at 10600 xg to

wash the spin column, and again the flow-through was discarded. The RNeasy Mini

spin column was placed in a new 1.5 ml collection tube and 50 µl of RNeasy-free water

was added, and centrifuged for 1 minute at 10600 xg to elute the RNA.

2.10.2 Analysis of purified RNA

The total purified RNA was separated using a 1% (w/v) agarose gel in 1 × MOPS

(morpholinopropanesulfonic acid) buffer. The MOPS stock buffer (10 × MOPS)

contained: 50 mM sodium acetate, 200 mM MOPS, and EDTA at pH 7.0. The RNA

loading buffer consisting of 50% (v/v) formamide, 16% (v/v) formaldehyde, 10% (v/v)

of 10 × MOPS buffer, 0.1 mg/ml ethidium bromide and 0.01% (w/v) bromophenol blue,

was mixed with the purified RNA, denatured at 70°C for 10 minutes, placed on ice for 2

minutes, and then the total RNA was separated by electrophoresis at 80 V for 1 hour.


59

2.10.3 Reverse transcriptase PCR

RNA was prepared as described in section 2.10.1 and used for cDNA synthesis and then

PCR amplification of two fragments, first one between glpX and hprK genes and second

between a gene that encodes for 5-formyltetrahydrofolate cyclo-ligase and hprK gene

using a QIAGEN® One-Step RT-PCR Kit (Cat# 210210). This kit contains a special

blended enzyme mix for both reverse transcription and PCR amplification. Omniscript

and Sensiscript Reverse Transcriptases are responsible for generating cDNA from the

RNA. Omniscript Reverse Transcriptase is specially engineered for reverse

transcription of RNA amounts greater than 50 ng. However, Sensiscript Reverse

Transcriptase is designed for use with very small amounts of RNA, less than 50 ng.

HotStarTaq DNA Polymerase is needed to generate the PCR products from the cDNA

and this enzyme does not interfere with the reverse-transcriptase reaction due to it only

being activated when the enzyme is heated to 95°C for 15 minutes. Details of the PCR

reaction mix and PCR cycles are presented in Table 2.13 and Table 2.14 respectively.

Table ‎2.13: Reaction mix for one-step RT-PCR


5x QIAGEN OneStep RT-PCR Buffer 10

dNTP Mix (containing 10 mM of each dNTP) 2

100 pmol/μl RNAglpX or RNAhprK forward

primer

1

100 pmol/μl RNAglpX or RNAhprK reverse

primer

1

QIAGEN OneStep RT-PCR Enzyme Mix 2

(4 U/μl) RNase inhibitor 2

Template RNA Variable

RNase-free water Variable

Total volume 50


60

Table ‎2.14: RT-PCR Cycling steps (QIAGEN® One-Step RT-PCR Kit)

Cycling step Time (min) Temperature (°C)

1-Reverse transcription step 30 50

2-Initial PCR activation step 15 95

3-Denaturation step 1 94

4-Annealing step 1 Variable

5-Primers extension step 1 72

Repeats steps 3 to 5 for 40 cycles

6-Final extension step 10 72

Also, the One Step RT-PCR Master Mix Kit from Novagen® was used to generate

cDNA and then PCR product. This kit utilizes an engineered Thermus thermophilus

(rTth) DNA polymerase, which acts both as reverse transcriptase and DNA polymerase.

Details of the PCR reaction mix and PCR cycles are presented in Table 2.15 and Table

2.16 respectively.

Table ‎2.15: Reaction mix for one-step RT-PCR


2X One Step RT-PCR Master Mix 25

50 mM Mn(OAc)2 2.5

10 pmol/μl RNAglpX or RNAhprK

forward primer

1

10 pmol/μl RNAglpX or RNAhprK

reverse primer

1

Template RNA Variable

RNAse-free water Variable

Total volume 50


61

Table ‎2.16: RT-PCR Cycling steps (One Step RT-PCR Master Mix Kit Novagen®

)

Cycling step Time Temperature (°C)

1-Enzyme Activation step 30 s 90

2-Reverse transcription step 30 min 60

3-Denaturation step (enzyme) 1 min 94

4-Denaturation step (DNA) 30 s 94

5-Annealing step 30 s variable

6-Extension step 1 min 72

Repeat steps 4–6 for 40 cycles

7-Final Extension 7 min 60

2.11 Proteomic analysis

A sample of crude extract of C. acetobutylicum grown on glucose minimal medium was

obtained from Dr. Wilfrid J Mitchell and sent to Moredun Research Institute by Dr.

Douglas Fraser-Pitt. The proteomic analysis was carried out by using liquid

chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS).

2.12 Bioinformatics analysis

The National Centre for Biotechnology Information (NCBI) database was used to obtain

the DNA and amino acid sequence of each gene and protein respectively:

http://www.ncbi.nlm.nih.gov/.

Also, the Basic Local Alignment Search Tool (BLAST), which is one of the search

tools in NCBI database, was used to determine the percent homology in sequence

alignments:

http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome.

ClustalW2 on The European Molecular Biology Laboratory- European Bioinformatics

Institute (EMBL-EBI) database was used to align multiple DNA or protein sequences

and visualised via the programmes GeneDoc or TreeView:

http://www.ebi.ac.uk/Tools/msa/clustalw2/.

http://www.ncbi.nlm.nih.gov/

http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome

http://www.ebi.ac.uk/Tools/msa/clustalw2/

Characterisation of glpX gene

62

Chapter 3

3 Characterisation of glpX gene


63

3.1 Introduction

FBPase is one of the main irreversible gluconeogenic enzymes and is involved in

converting fructose 1,6-bisphosphate to fructose 6-phosphate and orthophosphate. This

protein has been characterised in many Gram positive and Gram negative bacteria such

as B. subtilis and E. coli (Brown et al., 2009; Jules et al., 2009). To date, there is no

study reporting FBPase activity in the genus Clostridium. Tangney et al, (2003)

reported that there is an open reading frame that might encode for a FBPase enzyme in

C. acetobutylicum ATCC 824. Therefore, the aim of this chapter was to examine

whether the open reading frame that has been reported does encode for FBPase and also

to examine whether there is any other gene that might encode for FBPase enzyme. This

was investigated by bioinformatic analysis, genetic complementation and biochemical

analysis (enzyme assay).

3.2 Results

3.2.1 Bioinformatic analysis

The whole genome of C. acetobutylicum ATCC 824 has been sequenced and is

accessible for analysis (Nölling et al., 2001; GenBank accession number AE001437

http://www.ncbi.nlm.nih.gov/nuccore/AE001437). C. acetobutylicum like all other

bacteria requires fructose-1,6-bisphosphatase activity to enable the organism to grow

under gluconeogenic conditions. The genome sequence of C. acetobutylicum was

searched for potential homologues of fructose-1,6-bisphosphatase encoding genes using

the NCIB database. Two potential homologues were identified, cac1088 and cac1572.

Phylogenetic analysis of cac1088 and cac1572 with both experimentally proven and

putative FPBase proteins from diverse species including human, yeast, plants and

bacteria revealed that cac1088 clusters with FBPase class II proteins (Figure 3.1). It

appears that cac1088 is evolutionarily divergent from E. coli FBPase class II.

Interestingly, cac1572 clusters with FBPase class III enzymes which including B.

subtilis FBPase enzyme (Fujita et al., 1998).

http://www.ncbi.nlm.nih.gov/nuccore/AE001437


64

Figure ‎3.1: Unrooted phylogenetic tree of characterised FBPases and the two putative clostridial FBPases

Class I

Class IIClass III


65

Continuation of Figure 3.1. Abbreviations for FBPases Class I: b.na fbp; Brassica

napus (Rape), s.po fbp; Schizosaccharomyces pombe (Fission yeast), h.sa fbp; Homo

sapiens (Human), e.co fbp; Escherichia coli fbp, s.el_fbp; Synechococcus elongatus

PCC 7942 and p.sa fbp: Pisum sativum (Garden pea). For FBPases Class II: s.el glpX;

Synechococcus elongatus PCC 7942, c.gl glpX; Corynebacterium glutamicum, m.tu

glpX; Mycobacterium tuberculosis, e.co glpX; Escherichia coli, cac1088; C.

acetobutylicum glpX, b.su glpX; Bacillus subtilis. For FBPases Class III: cac1572; C.

acetobutylicum fbp, b.su fbp; Bacillus subtilis, s.au fbp; Staphylococcus aureus.

3.2.2 The cac1088 gene

The first gene identified cac1088, covers 975 bp and putatively encodes a class II GlpX-

like protein of 324 aa. The estimated molecular weight of the protein is 34.81 kDa. A

BLAST search was conducted on the NCIB protein database using the deduced amino

acid sequence of the putative GlpX protein; the results are presented in Table 3.1. All

of the top ten hits from the BLAST search were from the genus Clostridium. The

putative FBPase of C. botulinum A str. ATCC 3502 shares 78% identity with the

putative C. acetobutylicum FBPase protein (cac1088). The cac1088 also shares

similarity with putative FBPase of other Clostridium spp.

The protein sequences of the top ten BLAST hits were then aligned in ClustalW2

(www.ebi.ac.uk/Tools/msa/clustalw2/) together with the experimentally proven type II

FBPase protein sequences such as E. coli and M. tuberculosis and then visualised in

GeneDoc (www.nrbsc.org/gfx/genedoc/). There are large areas of conservation

throughout the length of the proteins (Figure ‎3.2). There are four blocks of conserved

motifs (Brown et al., 2009) consisting of VIGEGE, APML, AVDPIDGT and DGDV in

sequence block I, II, III and IV respectively (Figure ‎3.2). The amino acid residues

AVDPIDGT conserved in motif III have been shown to be involved in metal ion

binding and phosphatase catalytic activities by crystallographic and mutational analysis

(Brown et al., 2009). It is interesting to note that the presence of the conserved block

III in proteins is not sufficient for identification of FBPase members (Brown et al.,

2009; York et al., 1995).

http://www.nrbsc.org/gfx/genedoc/


66

Table ‎3.1: BLAST homology results for the deduced amino acid sequence of the

putative fructose 1,6-bisphosphatase II (cac1088).

Species

Putative

protein % Identity % Similarity Predicted

molecular

mass (Da)

Accession number

C.

carboxidivorans

P7

Fructose 1,6-

bisphosphatase

, class II

78

89 34364

ZP_05392

875

C. sporogenes

ATCC 15579

Fructose 1,6-

bisphosphatase

, class II

78 89 34306 ZP_02995

269

C. botulinum A

str. ATCC 3502

Fructose 1,6-

bisphosphatase

, class II

78

89

34304

YP_00125

5113

C. botulinum A3

str. Loch Maree

Fructose 1,6-

bisphosphatase

II

77

89

34304

YP_00178

7935

C. botulinum

B1 str. Okra

Fructose 1,6-

bisphosphatase

II

77

89

34286

YP_00178

2231

C. novyi NT

Fructose 1,6-

bisphosphatase

II

77

89

34227

YP_87773

6

C. botulinum C

str. Eklund

Fructose 1,6-

bisphosphatase

, class II

76

90

34281

ZP_02621

295

C. botulinum D

str. 1873

Fructose 1,6-

bisphosphatase

, class II

75

89

34216

ZP_04862

266

C. botulinum

BKT015925

Fructose 1,6-

bisphosphatase

II

75

89

34255

YP_00439

5515

C. tetani E88

Fructose 1,6-

bisphosphatase

II

74

87

34339

NP_78165

7


67

Continuation of Figure 3.2

C.ace :

C.boA :

C.bo_BKT :

C.bo_B1 :

C.bo3 :

C.sp :

C.nov :

C.bo_C :

C.bo_D :

C.car :

C.tet :

Mtb :

Eco :

* 20 * 40 *

---------------MFDNDISMSLVRVTEAAALQSSKYMGRGDKIGADQAAVDG

---------------MLNTDIAMGLARVTEAAALSASKFMGRGDKNAADQAAVDG

---------------MKNLDVSMGLVRVTEAAALNGAKLMGRGDKNAADQAAVDG




---------------MNNLDVSMGLVRVTEAAALNGAKLMGRGDKNAADQEAVNG

---------------MNNLDVSMGLIRVTEAAALNGSKLMGRGDKNAADQEAVNG

---------------MKNLDVSMGLVRVTEAAALNGAKLMGRGDKNAADQAAVDG

---------------MLNTDIAMGLARVTEAAALCSSKFMGRGDKIAADQAAVDG

---------------MIKQDIAMGLVRVTEAAALCSSKYMGRGDKIAADQAAVDG

MASHDPSHTRPSRREAPDRNLAMELVRVTEAGAMAAGRWVGRGDKEGGDGAAVDA

----------------MRRELAIEFSRVTESAALAGYKWLGRGDKNTADGAAVNA

: 40

: 40

: 40

: 40

: 40

: 40

: 40

: 40

: 40

: 40

: 40

: 55

: 39

C.ace :

C.boA :

C.bo_BKT :

C.bo_B1 :

C.bo3 :

C.sp :

C.nov :

C.bo_C :

C.bo_D :

C.car :

C.tet :

Mtb :

Eco :

60 * 80 * 100 *

MEKAFSFMPVRGQVVIGEGELDEAPMLYIGQKLGMGKDYMPEMDIAVDPLDGTIL

MHKAFQIMPIRGKVVIGEGELDEAPMLYIGEEVGIGAEDMVEMDIAVDPVDGTKL

MEKAFNMMPIRGKVVIGEGEMDEAPMLYIGQSVGVGEEKMPEMDIAVDPIDGTVL

MHKAFQIMPIRGKVVIGEGELDEAPMLYIGEEVGIGAEDMVEMDIAVDPVDGTKL

MHKAFQIMPIRGKVVIGEGELDEAPMLYIGEEVGIGAEDMLEMDIAVDPVDGTKL

MHKAFQIMPVRGKVVIGEGELDEAPMLYIGEEVGIGAEDMVEMDIAVDPVDGTKL

MEKAFNMMPIRGNVVIGEGEMDEAPMLYIGQKVGVGQEDMPEMDIAVDPIDGTVL

MEKAFNMMPIRGNVVIGEGEMDEAPMLYIGQKVGIGREDMPEMDIAVDPIDGTVL

MEKAFNMMPIRGRVVIGEGEMDEAPMLYIGQNVGVGEENMPEMDIAVDPIDGTVL

MEKAFAMMPVRGTVVIGEGELDNAPMLYIGQSVGVGSADMPEMDIAVDPLDGTIL

MEKAFSMLPIKGTVVIGEGEMDEAPMLYIGQKLGIGTDGMEEMDIAVDPLDGTVL

MRELVNSVSMRGVVVIGEGEKDHAPMLYNGEEVGNG--DGPECDFAVDPIDGTTL

MRIMLNQVNIDGTIVIGEGEIDEAPMLYIGEKVGTG--RGDAVDIAVDPIEGTRM

: 95

: 95

: 95

: 95

: 95

: 95

: 95

: 95

: 95

: 95

: 95

: 108

: 92

C.ace :

C.boA :

C.bo_BKT :

C.bo_B1 :

C.bo3 :

C.sp :

C.nov :

C.bo_C :

C.bo_D :

C.car :

C.tet :

Mtb :

Eco :

120 * 140 * 160

ISKGLPNAIAVIAMGPKGSLLH-APDMYMKKIVVGPGAKGAIDINKSPEENILNV

IAKGLENAIAVVAMGPKGSLFH-APDMYMKKIAVGAGAKGAIDINKSPKENILNV

IAKGLPNSIAVVAMGPKGSLLH-APDMYMQKIAVGPGARGSIDINKSPRENIINV



IAKGLENAIAVVAMGPKGSLFH-APDMYMKKIAVGSGAKGAIDINKSPKENILNV

IAKGLPNSIAVVAMGPKGSLLH-APDMYMKKIAVGPGAVGAIDINKSPKENIINV

IAKGLPNSIAVVAMGPKGSLLH-APDMYMKKIAVGPGAKGAIDINKSPKENIINV

IAKGLPNSIAVVAMGPKGSLLH-APDMYMQKIAVGPGAKGAIDINKSPKENIINV

IAKGLPNAIAVVAMGPKESLFH-APDMYMKKIAVGPGAKGAIDINKSPKENIINV

IAKGLPNAVSVMAMGPKGSLLN-APDMYMKKIAVGASAKGVIDINKTNKENIINV

MSKGMTNAISVLAVADRGTMFDPSAVFYMNKIAVGPDAAHVLDITAPISENIRAV

TAMGQANALAVLAVGDKGCFLN-APDMYMEKLIVGPGAKGTIDLNLPLADNLRNV

: 149

: 149

: 149

: 149

: 149

: 149

: 149

: 149

: 149

: 149

: 149

: 163

: 146

C.ace :

C.boA :

C.bo_BKT :

C.bo_B1 :

C.bo3 :

C.sp :

C.nov :

C.bo_C :

C.bo_D :

C.car :

C.tet :

Mtb :

Eco :

* 180 * 200 * 220

AKALNKDISELTVIVQERERHDYIVKAAIEVGARVKLFGEGDVAAALACGFEDTG

ARALNKDVTELTVIVQERDRHDYIVKDAREVGARVKLFGEGDVAAVLACGFEDTG

AKALNKDIEDLTVIVQERERHNYIVEQAREVGARVKLFAEGDVAAILACGFENTG




AKALNKDIEDLTIIVQERERHDYIVEQAREVGARVKLFGEGDVAAILACGFENTG

AKALSKEIEDLTVIVQERERHDYIVEQAREVGARVKLFGEGDVAAILACGFENTG

AKALNKDIEDLTVIVQERERHNYIVEQAREVGARVKLFAEGDVAAILACGFENTG

SKALNKDITEMTVIVQERERHNYIVQAAREVGARVKLFGEGDVAAVLACGFENTG

AMALNKDVTELTIMVQERERHNDIIKDAREVGARVKLFSEGDVAAVLACGFESTG

AKVKDLSVRDMTVCILDRPRHAQLIHDVRATGARIRLITDGDVAGAISACRPHSG

AAALGKPLSELTVTILAKPRHDAVIAEMQQLGVRVFAIPDGDVAASILTCMPDSE

: 204

: 204

: 204

: 204

: 204

: 204

: 204

: 204

: 204

: 204

: 204

: 218

: 201

I

IV

II III


68

Figure ‎3.2: Alignment of the putative C. acetobutylicum fructose 1,6-

bisphosphatase II (cac1088) sequence with the closest homologues.

The deduced amino acid sequence of the putative C. acetobutylicum fructose 1,6-

bisphosphatase II protein is aligned with the fructose 1,6-bisphosphatase protein

sequences with the highest percentage identities from the BLAST output and

experimentally verified FBPase II enzymes in M. tuberculosis (Mtb) and E. coli (Eco).

Dashes within the sequences indicate gaps giving optimal alignment. The amino acids

are highlighted according to 90% or more (red), 80% (yellow), 60% (grey) and non-

highlighted less than 60% conservation. Abbreviations: C.ace, C. acetobutylicum ATCC

824;

C.ace :

C.boA :

C.bo_BKT :

C.bo_B1 :

C.bo3 :

C.sp :

C.nov :

C.bo_C :

C.bo_D :

C.car :

C.tet :

Mtb :

Eco :

* 240 * 260 *

IDILMGIGGAPEGVIAAAAIKCMGGEMQAQLIPHTQE-------------EIDRC

VDIMMGTGGAPEGVIAAAAIKCMGGEMQAQLCPTSQE-------------EIERC

IDLMIGTGGAPEGVIAAAAIKCMGGEIQAKLEPHTDE-------------ERERC

VDIMMGTGGAPEGVIAAAAIKCMGGEIQAQLCPTSQE-------------EIERC

VDVMMGTGGAPEGVIAAAAIKCMGGEMQAQLCPTSQE-------------EIERC

VDIMMGTGGAPEGVIAAAAIKCMGGEMQAQLCPTSQE-------------EIERC

IDLMIGTGGAPEGVIAAAAIKCMGGEMQAILEPHTEE-------------EIKRC

IDLMIGTGGAPEGVIAAAAIKCMGGEIQAILEPHTEE-------------EVRRC

IDLMIGTGGAPEGVIAAAAIKCMGGEIQAKLEPHTDE-------------ERERC

VDIFMGTGGAPEGVIAAAAIKCMGGDMQAKLEPHTDE-------------ERERC

VDMMMGTGGAPEGVIAAAAIKCMGGDMQAKLVPQNEE-------------EIRRC

TDLLAGIGGTPEGIIAAAAIRCMGGAIQAQLAPRDDA-------------ERRKA

VDVLYGIGGAPEGVVSAAVIRALDGDMNGRLLARHDVKGDNEENRRIGEQELARC

: 246

: 246

: 246

: 246

: 246

: 246

: 246

: 246

: 246

: 246

: 246

: 260

: 256

C.ace :

C.boA :

C.bo_BKT :

C.bo_B1 :

C.bo3 :

C.sp :

C.nov :

C.bo_C :

C.bo_D :

C.car :

C.tet :

Mtb :

Eco :

280 * 300 * 320 *

HKMGIDDVNKIFMIDDLVKSDNVFFAATAITECDLLKGIVFSKNERAKTHSILMR

KTMGIKDHEAILYIDDLVKSDDVYFAATAITDCDLLKGVVYNRNEKAITNSIVMR

KLMGIEDIEKVLTMDDLVMSDDVYFAATALTDSDLLKGVIFSKGDVATTHSVVMR




KEMGIEDINKVLTTDDLVKSDDVYFAATALTDSDLLKGVVFSKNDMATTHSVVMR

KDMGIEDINKVLTMDDLVKSDDVYFAATALTDSDLLKGVVFSKNDVATTHSVVMR

KLMGIEDVEKVLTMDELVMSDDVHFAATALTDSDLLKGVVFSKGDMATTHSVVMR

KSMGISDLNKVLLINDLVKDDEVYFAATGITDCDLLRGVVFSKNDWATTHSVVMR

KSMGIENVEATLYINDLVKSDDIYFAATAVTDSDLLKGVVYSKNNWATTQSIVMR

LEAG-YDLNQVLTTEDLVSGENVFFCATGVTDGDLLKGVRYYPGG-CTTHSIVMR

KAMG-IEAGKVLRLGDMARSDNVIFSATGITKGDLLEGIS-RKGNIATTETLLIR

: 301

: 301

: 301

: 301

: 301

: 301

: 301

: 301

: 301

: 301

: 301

: 313

: 309

C.ace :

C.boA :

C.bo_BKT :

C.bo_B1 :

C.bo3 :

C.sp :

C.nov :

C.bo_C :

C.bo_D :

C.car :

C.tet :

Mtb :

Eco :

340 * 360

SKTGTIRFVEAIHDLNK------------SKLVVE--

SKTGTIRFVKACHNLAK------------SAIVVE--

SKTRTIRFVEAVHCAEK------------SCLL----







AKTGTVRFIEAHHDLKR------------SSLVVR--

SKTGTIRFVDAQHHLSR------------SNLVL---

SKSGTVRMIEAYHRLSKLNEYSAIDFTGDSSAVYPLP

GKSRTIRRIQSIHYLDR----------KDPEMQVHIL

: 324

: 324

: 322

: 324

: 324

: 324

: 322

: 322

: 322

: 324

: 323

: 350

: 336


69

C.car, C. carboxidivorans P7; C.sp, C. sporogenes ATCC 15579; C.boA, C. botulinum

A str. ATCC 3502; C.bo3, C. botulinum A3 str. Loch Maree; C.bo B1, Clostridium

botulinum B1 str. Okra; C.nov, C. novyi NT; C.bo C, C. botulinum C str. Eklund; C.bo

D, C. botulinum D str. 1873; C.bo BKT, C. botulinum BKT015925; C.tet, C. tetani E88;

Mtb, M. tuberculosis and Eco, E. coli.

3.2.3 The cac1572 gene

Bioinformatic analysis of the locus tag cac1572 of the C. acetobulycum genome

revealed the presence of a second putative fructose 1,6-bisphosphatase-like class III

protein (Fbp) of 665 aa. A BLAST search using the deduced amino acid sequence of the

putative C. acetobulycum Fbp protein revealed that cac1572 is closely related to

FBPase_2 family of proteins (Pfam06874). This family consists of several bacterial

fructose-1,6-bisphosphatase proteins (EC:3.1.3.11) belonging to the Firmicutes phylum.

The Pfam06874 appears to be not structurally related to class II FBPase_GlpX enzymes

(Pfam003320). Also, the BLAST search results are presented in Table 3.2. All of the

top ten hits from the BLAST search are from the genus Clostridium. The putative

FBPase of C. cellulovorans 743B shares 73% identity with the putative C.

acetobutylicum FBPase protein (cac1572). The cac1572 also shares similarity with

putative FBPases of other Clostridium spp. The protein sequences of the top ten

BLAST hits were then aligned in ClustalW2 together with the experimentally proven

class III FBPases protein sequences of B. subtilis (Fujita et al., 1998) and then

visualised in GeneDoc. Several conserved motifs can be seen through the alignment

result together with the B. subtilis enzyme indicating that putative C. acetobutylicum fbp

gene (cac1572) is most likely to encode for a class III FBPase protein.


70

Table ‎3.2 BLAST homology results for the deduced amino acid sequence of the

putative fructose 1,6-bisphosphatase III (cac1572).

Species Putative

protein % Identity % Similarity Predicted

molecular

mass (Da)

Accession number

C.

cellulovorans

743B

Fructose 1,6-

bisphosphatase

73

87

76707

YP_00384

2980

C. beijerinckii

NCIMB 8052

Fructose 1,6-

bisphosphatase

73

87

76726

YP_00130

9581

C. botulinum D

str. 1873

Fructose 1,6-

bisphosphatase

71

83

76535

ZP_04863

125

C. botulinum C

str. Eklund

Fructose 1,6-

bisphosphatase

70

84

76294

ZP_02620

031

C. botulinum

BKT015925

Fructose 1,6-

bisphosphatase

70

83

76412

YP_00439

5366

C. novyi NT

Fructose 1,6-

bisphosphatase

68

83

76286

YP_87759

3

C. ljungdahlii

DSM 13528

Fructose 1,6-

bisphosphatase

68

83

77329

YP_00378

1055

C. botulinum

A2 str. Kyoto

Fructose 1,6-

bisphosphatase

69

85

77256

YP_00280

2849

C. botulinum Bf

Fructose 1,6-

bisphosphatase

69

85

77257

ZP_02616

118

C. sporogenes

ATCC 15579

hypothetical

protein

68

85

77298

ZP_02994

084


71


C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

* 20 * 40 *

------MLLESNTKNEEIKD-----------NLKYLVLLSKQYPTINEAATEIINLQA

------MTFCDENNLDLIKR-----------DLRYLNLLSKQYPDISSASTEIVNLQA

------MTFCDENNLDLIKR-----------DLRYLKLLAKQYPDISSASTEIVNLQA

------MTFCDENNLDLIKK-----------DLRYLKLLANQYPNISSASTEIVNLQA

------MTFCDENNLDLIKK-----------DLRYLKLLANQYPNISSASTEIVNLEA

------MIFYDENNLDIIEK-----------DLRYLQLLSREYPTISSASTEIINLQA

------MTLYDENNLHIIKD-----------NLRYLKLLSKQYPSISSASSEIINLQA

------MTLYDENNLHIIKD-----------NLRYLKLLSKQYPSISSASSEIINLQA

------MSIHKELNELDVEK-----------DLRYLNLLSKEYSTINSVCTEFINLQS

-------MKKYDLSTNEISD-----------NLRYLELLSKQYPTINEASTEIINLQA

MFKNNVILLNSPYHAHAHKEGFILKRGWTVLESKYLDLLAQKYDCEEKVVTEIINLKA

: 41

: 41

: 41

: 41

: 41

: 41

: 41

: 41

: 41

: 40

: 58

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

60 * 80 * 100 *

ILNLPKGTEHFLSDVHGEYEQFIHVLKNASGVIKRKIDDIFGNRLMQSEKKSLATLIY

ILNLPKSTEHFLSDIHGEYESFTHVLKNASGVIKRKIDDVFGNSLRDSEKLTLATVIY



ILNLPKSTEHFLSDIHGEYESFTHVLKNASGVIKRKIDDVFGNSLRDNEKLTLATVIY

ILNLPKATEHFISDIHGEYESFTHMLRNASGVIKRKIDDVFGNSLREEQKASLATLVY

ILNLPKGTEHFISDVHGEYESFTHMLKNASGVIKRKIDDVFGTSLRECDKKNLATLIY

ILNLPKGTEHFISDVHGEYESFTHMLKNASGVIKRKIDDVFGTSLRECDKKNLATLIY

ILNLPKGTEHFLTDIHGEYEQFNHVLKNASGVIKRKIEDIFGNTLRQNEKKSLATLIY

ILNLPKGTEHFLTDIHGEYEPFIHVLKNASGVIKRKIEDLFGNSLMQSEKKSLATLIY

ILNLPKGTEHFVSDLHGEYQAFQHVLRNGSGRVKEKIRDIFSGVIYDREIDELAALVY

: 99

: 99

: 99

: 99

: 99

: 99

: 99

: 99

: 99

: 98

: 116

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

120 * 140 * 160 *

YPEQKLDIILK---QEKNIDDWYKITLYRLIEVCRNVSSKYTRSKVRKALPKEFSYII

YPEQKLELIKQ---SEKDLSDWYKITLYRLIELCRVVSSKYTRSKVRKALPHDFAYII

YPEQKLELIKQ---CEKDLSDWYKITLYRLIELCRAVSSKYTRSKVRKALPHDFAYII



YPERKIELIKE---KEHNLEEWYRISLYQLIELCKNVSSKYTRSKVRKALPSDFSYII

YPEQKLDLIKK---SEKNLEDWYKITLYRLIRLCQIVSSKYTRSKVRKSLPSDFAYII

YPEQKLDLIKK---SEKNLEDWYKITLYRLIRLCQIVSSKYTRSKVRKSLPSDFAYII

YPEQKLDIVLK---EEENINDWYTITLYRLIEICRHVSSKYTRSKVRKALPKDFAYII

YPEQKLEIVLK---QEENIDDWYKINLYRLIEICRYVSSKYTRSKVRKALPKDFTYII

YPEDKLKLIKHDFDAKEALNEWYKETIHRMIKLVSYCSSKYTRSKLRKALPAQFAYIT

: 154

: 154

: 154

: 154

: 154

: 154

: 154

: 154

: 154

: 153

: 174

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

180 * 200 * 220 *

EELLHEQPKGVDKQEYYDEIIKTIISIDRAKEFITAISKLIQRLVVDRLHIIGDIFDR

EELLHEHDGAFNKQEYYNGIVSTIIDIDRAPEFITAISKVIQRLVVDRLHIIGDIYDR

EELLHEHDGTINKQEYYNGIISTIIDIDRAPEFITAISKVIQRLVVDRLHIIGDIYDR

EELLHEHDGTINKHEYYNGIISTIIDIDRASEFITAISKVIQRLVVDRLHIIGDIYDR

EELLHEHDGTINKHEYYNGIVSTIIDIDRAPEFITAISKVIQRLVVDRLHIIGDIYDR

EELLHDQDDKVDKQAYYNGIIKTIIDINRAPEFIIAISNVIQRLVVDRLHIIGDIYDR

EELLNEQGDRVDKQEYYNSIIETIIDIDRASEFIIAISNVIQRLVVDKLHIIGDIYDR

EELLNEQGDRVDKQEYYNSIIETIIDIDRASEFIIAISNVIQRLVVDKLHIIGDIYDR

EELLHEYPESLDKHGYYGEIVKTIVAIDRAREFIMALSNLIQRLVIDRLHIIGDIFDR

EELLHEQPKGIDKYEYYEQIIRTIIDTDRSKEFIVALSKLIQRLVIDRLHILGDIFDR

EELLYKTEQAGNKEQYYSEIIDQIIELGQADKLITGLAYSVQRLVVDHLHVVGDIYDR

: 212

: 212

: 212

: 212

: 212

: 212

: 212

: 212

: 212

: 211

: 232

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

240 * 260 * 280 *

GPRADIIMDKLEEYHAVDIQWGNHDILWMGAASGSSVCMANVIRISARYANLSTIEDG

GPGAEIILEALMKHHSVDIQWGNHDILWMGAAAGSEACICNVLRISLRYANLNTIEDG

GPGAEIILEALMKHHSVDIQWGNHDILWMGAAAGSEACICNVLRISLRYANLNTIEDG

GPGAEIIMEELMKHHSVDIQWGNHDILWMGAAAGSEACICNVLRISLRYANLNTIEDG

GPGAEIIMEELMKHHSVDIQWGNHDILWMGAAAGSEACICNVLRISLRYANLNTIEDG

GPGAEIIMDALMKHHSVDIQWGNHDMLWMGAAAGCEACIANVLRISLRYANLHTIEDG

GPGAEIIIEALSKHHSIDIQWGNHDIVWMGAAAGCEACIANVIRISLRYANLSTLEDG

GPGAEIIIEALSKHHSIDIQWGNHDIVWMGAAAGCEACIANVIRISLRYANLSTLEDG

GPGAEIILDTLKNYHSVDIQWGNHDVLWMGACAGSKACIANVIRISARYSNLDTIEDG

GPGADIIMDTLVEYHSVDIQWGNHDILWMGAACGSDVCIANVIKNSLKYANLDTLENG

GPQPDRIMEELINYHSVDIQWGNHDVLWIGAYSGSKVCLANIIRICARYDNLDIIEDV

: 270

: 270

: 270

: 270

: 270

: 270

: 270

: 270

: 270

: 269

: 290


72

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

300 * 320 * 340

YGINLLPLATFAMDFYGNDKCKNFEPKIESDKSYTVKEIELIGKMHKAIAIIQFKLEG

YGINLLPLATFAMDIYENDPCNSFIPKT-INKELTQNEINLISKMHKAIAIIQFKLQG

YGINLLPLATFAMDIYENDPCNSFIPKT-INKELTQNEINLISKMHKAISIIQFKLQG

YGINLLPLATFAMDVYENDPCNSFIPKT-INKELTQNELNLISKMHKAIAIIQFKLQG

YGINLLPLATFAMDVYENDPCNSFIPKT-INKELTQNELNLISKMHKAIAIIQFKLQG

YGINLLPLATFALEFYKDDPCKNFIPKT-LNKDISKNELNLLAKMHKAIAIMQFKLEA

YGINLLPLATFAMDFYKEDNCENFKPRT-IDKNLNETDIKLLSKMHKAISIIQFKLEG

YGINLLPLATFAMDFYKEDNCENFKPRT-IDKNLNETDIKLLSKMHKAISIIQFKLEG

YGINLLPLATFALDFYKEDPCTGFFPKFDAEKNYSVTEIELIAKMHKAIAIIQFKLEG

YGINLLPLATFSMDFYKDHPCNIFLPKMDCDKKYSINEINLIAQMHKAIAIILFKLEG

YGINLRPLLNLAEKYYDDNP--AFRPKA--DENRPEDEIKQITKIHQAIAMIQFKLES

: 328

: 327

: 327

: 327

: 327

: 327

: 327

: 327

: 328

: 327

: 344

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

* 360 * 380 * 400

EAIKRHPEFKMEHRMLLNKINFEDSTIELDGKKYKLNDTSFPTIDKNDPYKLIDEERE

QIIKNHPEFKMDDQLLLDKINYEKGTINLDGNVYKLNDTIFPTINPKDPYTLSPREND

QIIKNHPEFKMDDQLLLDKINYEKGTISLDGNIYKLNDTVFPTIDPKNPYTLTPGEDD

QIIQNHSEFKMDDQLLLDKIDYEKGTINLSGKIYKLNDTYFPTIDPKNPYKLTESEDD

QIIKNHPEFKMDDQLLLDKINYEKGTIDLDGHIYKLNDTFFPTVDPKDPYKLTENEDD

EIIKRHPEFKMDDRLLLDKIDIEKWEIDIYGHNYKLNDSKFPTIDWNDPYKLTDREQE

KIIKRRPEFKMEERLLLDKINIKEGTLNLNEKIYKLIDTNFPTLDKENPYELNERERD

KIIKRRPEFKMEERLLLDKINIKEGTLNLNEKIYKLIDTNFPTLDKENPYELNERERD

EIINRRPGFNMDDRLLLNKINFEAGTIELDGKTYKLNDTYFPTIDPKDPYKLLKEEQE

QVILRHPEFNMNHRLLLNKINYAEGTINLNGKTHKLKDSFFPTIDPKNPYELTYDEKE

PIIKRRPNFNMEERLLLEKIDYDKNEITLNGKTYQLENTCFATINPEQPDQLLEEEAE

: 386

: 385

: 385

: 385

: 385

: 385

: 385

: 385

: 386

: 385

: 402

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

* 420 * 440 * 460

VVEKLRSSFVNSEKLNRHVRFLFSHGNLYLKFNSNLLYHGCIPLNEDGTFKEVLIGSH

LIRKLTKSFVNSEKLQRHIRFLYSKGSMYLIYNSNLLYHGCIPLNEDGTFKEVTIDGK

LIKKITKSFVNSEKLQRHIRFLYSKGSMYLIYNSNLLYHGCIPLNEDGTFKEVTIDGK

LIKKLARSFVNSEKLQRHIRFIYSKGSMYLVYNSNLLYHGCIPLNEDGTFREISINGT

LIKKLTRSFVNSEKLQRHIRFMYSKGSMYLVYNSNLLYHGCVPLNEDGTFKEITIDGV

LIEKLTSSFVNSEKLQRHIKFLYGKGSIYLVYNSNLLYHGCIPLNKDGSFREVKIGKI

LVEKLTNSFINSEKLQRHIKFLYSNGSLYLKYNSNLLYHGCIPLNEDGSLKEVTLCKE

LVEKLTNSFINSEKLQRHIKFLYSNGSLYLKYNSNLLYHGCIPLNEDGSLKEVTLCKE

LVEKLRSSFMNSDKLNKHVRFLFANGSIYLKYNSNLLYHGCIPMNENGTFKKVRLRDK

LIDKLKTSFINSDKYNKHVRFLYSNGSLYLKFNSNLLYHGFIPLNEDGSFKKVKIADK

VIDKLLFSVQHSEKLGRHMNFMMKKGSLYLKYNGNLLIHGCIPVDENGNMETMMIEDK

: 444

: 443

: 443

: 443

: 443

: 443

: 443

: 443

: 444

: 443

: 460

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

* 480 * 500 * 520

KYKGKALLDKLDVLARKSFFYEENSKNSKYENDMIWYLWSGPFSPLFGKEKMTTFERY

KYWGKSLLDKFDCLAREAFFFKEDSKIKKFAMDMVWYLWCGPNSPEFGKYRMTTFERY

KYFGKSLLDKFDCLAREAFFFKEDSRLKKFAMDMVWYLWCGPNSPEFGKYKMATFERY

RYKGKSLLDKFDSLARESFFFKKGSKAKKFALDMVWYLWCGPNSPEFGKLRMTTLERY

RYSGKSLLDKFDSLAREAFFFKNGSSAKRFALDMMWYLWCGPNSPEFGKLRMTTLERY

SYSGRELLDRYDRLARDAFFFKNNTNSKKYGMDMMWYMWCGEDSPVFGKVRMTTFERY

TLKGKSLLDKLDRLAREAYFFKKDPESKLYGMDMMWYLWCGPNSPLFGKKKMTTFERY

TLKGKSLLDKLDRLAREAYFFKKDPESKLYGMDMMWYLWCGPNSPLFGKKKMTTFERY

EYNGKSLLDRLEILAREGYFHEDNPAAKLFGTDIMWYLWTGPYSPLFGKEKMTTFERY

EYKGKELLDKLDMLAREAYFSKDKDDSDNK-KDIMWYLWCGASSPLFGKDRMTIFEQY

PYAGRELLDVFERFLREAFAHPEET--DDLATDMAWYLWTGEYSSLFGKRAMTTFERY

: 502

: 501

: 501

: 501

: 501

: 501

: 501

: 501

: 502

: 500

: 516

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

* 540 * 560 * 580

FIDDKKTHYEKKDPYYHYRDDEDICINILREFGLDSEQAHIINGHVPVESKNGENPIK

FIDNEGTHIEYRNPYYKYRSDEKIVINILKEFGLDPNCSHIINGHIPVKTKAGENPIK

FIDNEGTHIEYRNPYYKYRSNEKVVINILKEFGLDPNCSHIINGHIPVKTKAGENPIK

FIDDKGTHIEYRNPYYKYRSNEDVVINILKEFGLDPDCSHIINGHIPVKTKAGENPIK

FIDDKGTHIEYRNPYYKYRSNEKVVTNILKEFGLDPDCSHVINGHIPVKTKAGENPIK

FIDDKIAHYEKKNYYYQYRDDENVCKNILAEFNLPPESSHIINGHIPVKTKEGESPIK

FLDDKNTHKEQKNPYYKYRNDEKMCTMIFEEFELDADNSHIINGHIPVKTKEGENPIK

FLDDKNTHKEEKNPYYKYRNDEKMCTMIFEEFELDADNSHIINGHIPVKTKEGENPIK

FIDDKKTHIEKKDPYYNFRDKEEVYDNIFSEFGLDPNESHIVNGHVPVEKKKGESPIK

FIEEKETHYEKKDPYFSLRDNEDICKKILKEFGLSSPESHIINGHMPVEEKNGESPIK

FIKEKETHKEKKNPYYYLREDEATCRNILAEFGLNPDHGHIINGHTPVKEIEGEDPIK

: 560

: 559

: 559

: 559

: 559

: 559

: 559

: 559

: 560

: 558

: 574


73


Figure ‎3.3: Multiple alignment of the putative C. acetobutylicum fructose 1,6-

bisphosphatase (cac1572) sequence with the top 10 results from the BLAST search.

The deduced amino acid sequence of the putative C. acetobutylicum fructose 1,6-

bisphosphatase protein is aligned with the fructose 1,6-bisphosphatase protein

sequences with the highest percentage identities from the BLAST output. Dashes

within the sequences indicate gaps giving optimal alignment. The amino acids are

highlighted according to 90% or more (red), 80% (yellow), 60% (grey) and non-

highlighted less than 60% conservation. Abbreviations: C.acet, C. acetobutylicum

ATCC 824; C.cell, C. cellulovorans 743B; C.beij, C. beijerinckii NCIMB 8052; C.bot,

C. botulinum D str. 1873; C.bot C, C. botulinum C str. Eklund; C.bot BKT, C.

botulinum BKT015925; C.novy, Clostridium novyi NT; C.botA2, C. botulinum A2 str.

Kyoto; C.botB, C. botulinum Bf; C.ljun, C. ljungdahlii DSM 13528 and B.sub, B.

subtilis str. 168.

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

* 600 * 620 * 6

ANGKLIVIDGGFSKAYQSKTGIAGYTLIYNSFGLQLVSHELFETTEKAIKEETDIISS

ANGKLLVIDGGFCRAYQPETGIAGYTLIYNSYGLLLSSHEPFSSIRKAIEEEKDILSS


ANGKLLVIDGGFCRAYQPETGIAGYTLIYNSYGLLLSSHEPFSSIRKAIDEEKDILSS


ANGRLLVIDGGFCKAYQPETGIAGYTLIYNSYGLLLTSHEPFGAIKNAIEEDKDILSS

ANGKLLVIDGGFCKAYQPQTGIAGYTLIYNSYGLLLTSHEPFSSIHKAIVEGNDILSS

ANGKLLVIDGGFCKAYQPQTGIAGYTLIYNSYGLLLTSHEPFSSIHKAIVEGNDILSS

AKGKLLVIDGGFSKAYQGKTGIAGYTLIYNSFGLQLVSHEPFQSTEKAIKEETDILSS

ANGTLLVIDGGFSKAYQPKTGLAGYTLIYNSFGLQLVSHQPFESTEAAIKEETDILST

ANGKMIVIDGGFSKAYQSTTGIAGYTLLYNSYGMQLVAHKHFNSKAEVLSTGTDVLTV

: 618

: 617

: 617

: 617

: 617

: 617

: 617

: 617

: 618

: 616

: 632

C.acet :

C.bot :

C.bot_BKT :

C.bot_C :

C.novy :

C.ljun :

C.botA2 :

C.botBf :

C.cell :

C.beij :

B.sub :

40 * 660 * 680

TVIFEKSVRRKRVGDTDIGKDLKKQLYELNLLLLAYKKGLIKEFVKS----

TIILEQVVSRKRVADTDIGKALKKQIRELEMLLIAYRKGLIKEQTSKI---

TIILEQVVSRKRVSDTDIGKNLKKQIRELEMLLIAYRKGLIKEQNSEK---

TIILEQVVSRKRVADTDIGKDLKKQIEELEMLLIAYRKGLIKEQDSKS---

TIILEQVVSRKRVADTDIGKELKKQIAELKMLLIAYRKGLIKEQDSKS---

TVILEHVTKRKRVEDTDIGKDLTKQIADLEKLLIAYRKGLIKEDNNQIE--

TTILEHVSSRKRVLDTDSGEEIKKQIHDLEMLLVAYRKGLIKEENEANIRF

TTILEHVSSRKRVLDTDSGEEIKKQIHDLEMLLVAYRKGLIKEENEANIRF

TVILEHSMVRKKVADTDVGAILQEQIKNLKMLLIAYRTGLIKEKNSI----

TLLLEQVVNRKRVEDTDVGVTLKQQIDDLKMLLNAYRKGLIKQQNKI----

KRLVDKELERKKVKETNVGEELLQEVAILESLR-EYR--YMK---------

: 665

: 665

: 665

: 665

: 665

: 666

: 668

: 668

: 665

: 663

: 671


74

3.3 Cloning glpX and fbp genes using TOPO cloning system

3.3.1 Cloning of the C. acetobutylicum class II glpX gene

The putative class II glpX-like gene (cac1088) was amplified by PCR using the

TOPOglpX reverse and forward primers (Table ‎2.5: Primer details with annealing

temperture 52°C and the purified genomic DNA of C. acetobutylicum as template, to

produce a PCR product of about 1.1 kbp (Figure 3.1). This fragment which contains a

single deoxyadenosine (A) at the 3´ ends was then cloned into the pCR2.1-TOPO vector

(Appendix 7.4) that contains single 3´-thymidine (T) overhangs allowing the PCR

product to ligate easily with the vector. Also, this vector contained the lacZα gene

encoding for α part of beta-galactosidase, an enzyme that converts the colourless X-gal

(5-bromo-4-chloro-3-indolyl-[beta]-D-galactopyranoside) modified galactose sugar into

a coloured product (blue). In the pCR2.1-TOPO vector, the cloning site is located

within the lacZα gene (Appendix 7.4). When the foreign gene is cloned into that site,

this results in disturbing the lacZα gene which in turn cannot produce the beta-

galactosidase peptide. Therefore, the colonies that contain the foreign gene would be

white and this is the foundation of the blue/white screening technique. The recombinant

plasmids were transformed into two chemically competent strains of E. coli Mach1™

-

T1R and TOP10F`. The Mach1™

-T1R is a genetically modified E. coli strain which

harbours a gene (tonA) confering resistantce to T1 phage. However, TOP10F` is a

genetically modified E. coli strain which harbours the lacIq repressor gene, hence,

reduces the background expression of the cloned gene. Different amounts of both

transformed cells (25, 50, 75, 100, and 125 µl) were plated on five different LB plates

(+Amp) for plasmid selection and X-gal for white/blue screening. The efficiency of

transformation was poor and the plates had 1, 6, 14, 49, and 74 white colonies,

respectively. These colonies were transferred onto fresh plates and given reference

numbers. Six colonies were selected (five white and one blue) for PCR screening to

check whether these colonies contained the insert or not. PCR screening was carried

out using the same TOPOglpX reverse and forward primers and the DNA template

which was the recombinant plasmid extracted using the boiling method. Four colonies

out of 6 were positive (contained the cloned glpX gene), 3 were white colonies and 1

was blue (Figure 3.1). These colonies were named and numbered based on two factors;

competent cells in which the gene was transformed (M for Mach1™-T1R and T for

TOP10F`) and colony colour (W for white and B for blue).


75

Table ‎3.3: Locations of TOPOglpX primers on the sequence of cac1088 (glpX

gene).

Bases underlined represent the location of TOPOglpX primers. Bases highlighted in

green, red and gray represent the start codon, stop codon and the hindIII cutting site

respectively. Obtained from Genome Information Broker.

http://gib.genes.nig.ac.jp/single/index.php?spid=Cace_ATCC824

ACTTTAGGACACTATAGCGCATATATAAATTTTTATGGTTGCAATGTGTTTATAAACAAATGTTTAATAT

AATCAAGGAGGGAAATTCATGTTTGATAATGATATATCCATGAGTTTAGTAAGAGTAACAGAAGCAGCAG

CACTACAATCTTCAAAGTATATGGGAAGAGGAGATAAAATTGGAGCTGATCAAGCAGCAGTAGATGGAAT

GGAGAAGGCATTTAGTTTTATGCCAGTAAGAGGCCAGGTTGTAATAGGAGAGGGAGAACTTGATGAAGCT

CCTATGCTTTATATAGGTCAAAAGCTTGGTATGGGAAAAGACTATATGCCTGAAATGGATATAGCAGTAG

ATCCTTTAGATGGAACGATTTTAATTTCTAAGGGACTACCTAATGCAATAGCAGTAATAGCAATGGGACC

AAAAGGAAGTTTACTTCATGCCCCAGATATGTATATGAAGAAAATTGTTGTGGGACCTGGAGCAAAAGGT

GCTATAGATATAAATAAATCTCCTGAAGAGAATATTTTAAATGTAGCAAAGGCATTAAACAAGGACATAT

CTGAATTAACAGTTATAGTTCAAGAAAGAGAAAGACATGACTACATAGTAAAAGCAGCTATAGAAGTTGG

AGCAAGAGTTAAGCTATTTGGTGAGGGCGATGTTGCAGCTGCACTTGCTTGTGGTTTTGAAGATACTGGA

ATAGACATACTTATGGGAATTGGAGGAGCTCCAGAAGGAGTTATAGCCGCAGCAGCTATCAAGTGCATGG

GCGGAGAAATGCAGGCTCAGCTTATACCTCATACTCAGGAAGAAATAGATAGATGTCACAAAATGGGAAT

AGATGATGTAAATAAAATTTTCATGATAGATGATTTAGTTAAAAGTGATAATGTGTTTTTTGCAGCTACA

GCAATAACAGAATGTGATCTTCTTAAGGGCATAGTATTTTCTAAAAATGAACGTGCAAAAACCCATTCCA

TATTAATGAGATCTAAAACTGGTACAATAAGATTTGTTGAAGCTATTCATGACTTGAATAAAAGTAAATT

AGTGGTAGAATAAAA

TTAAAATGTTCGATTTACGGCGAAGGGGGATAGTAAATGCAGGTAA

TOPOglpX forward primer

TOPOglpX reverse primer



76

Therefore, for example the number one blue Mach1™

-T1R colony on the plate is

referenced as M1B. White and blue colonies were tested for the presence of the insert

using the same TOPOglpX primers which were used to amplify the gene from

chromosomal DNA of C. acetobutylicum. Also the blue colony, considered to be

lacking an insert, was tested as a control. Surprisingly, the blue colony was positive

(contained an insert) and was termed M1B. Moreover, the 3 white colonies, T1W, M1W,

and M2W were positive, while the other two white colonies, M3W and T2W were

negative (no insert).

Figure ‎3.4: Two agarose gels of PCR amplification and screeing of glpX gene.

(A) Amplification of C. acetobutylicum putative class II FBPase gene (glpX) using

TOPOglpX primers forward and reverse as indicated by lanes 2 and 3. (B) PCR

screening of 6 colonies as indicated by lane 2, T1W strain, lane 3, M1W strain, lane 4,

M2W, lane 5, M1B strain, lane 6, T2W strain, and lane 7, M3W strain. Lane 1,

represents molecular weight makers (hyperladder I) from Bioline.

1 2 3

1kbp_

(A)

1 2 3 4 5 6 7

1kbp_

(B)

10kbp_

0.2kbp_

10kbp_

0.2kbp_


77

3.3.2 Determination of the orientation of the insertion using PCR analysis

In the pCR2.1-TOPO vector, the cloned gene can be placed under the control of the lac

or T7 promoter (appendix ‎7.4). Therefore, the orientation of the insert needs to be

determined using PCR and restriction digest analysis. After testing for the presence of

the glpX gene, the orientation of the insert (glpX) was determined by PCR using one of

the TOPOglpX primers (forward or reverse) along with the T7 promoter primer. For a

gene under the control of the lac promoter, the result will be a fragment of about 1.2

kbp generated by the TOPOglpX forward and T7 promoter primers and there will be no

product from the TOPOglpX reverse and T7 promoter primers. However, for a gene

under the control of the T7 promoter, the result will be a fragment of about 1.2 kbp

generated by the TOPOglpX reverse and T7 promoter primers, with no product for the

TOPOglpX forward and T7 promoter primers (Figure 3.5). Only one from the 6

colonies tested gave a product by using the TOPOglpX forward and T7 promoter

primers and this colony was the blue one (M1B) suggesting that the glpX gene was

under the control of lac promoter, and hence, it was selected for further analysis.

Strains T1W, M1W, and M2W contained inserts under the control of the T7 promoter.

surprisingly, even though they were white colonies in the plate; M3W and T2W did not

give any product using either combination of the TOPOglpX forward and T7 promoter

primers or the TOPOglpX reverse and T7 promoter primers (Figure 3.6).


78

Figure ‎3.5: Illustration of the strategy to determine the orientation of the insert in

the pCR2.1-TOPO vector.

The TOPOglpX reverse, TOPOglpX forward and T7 promoter primers were used in this

strategy. (A) When the insert is under the control of lac promoter and (B) when the

insert is under the control of T7 promoter. P in blue indicates the location of the

promoter that controls expression of the PCR product.

PCR Product

P

TOPOglpX reverse primer

TOPOglpX forward primer T7 promoter primer

(B)

PCR ProductP

TOPOglpX forward primer

TOPOglpX reverse primer T7 promoter primer

(A)


79

Figure ‎3.6: Agarose gel of PCR analysis to to determine the orientation of the glpX

gene in the vector.

The lanes A show amplification of the insert under the control of T7 promoter using the

TOPOglpX reverse and T7 promoter primers. The lanes B show amplification of the

insert under the control of lac promoter using the TOPOglpX forward and T7 promoter

primers. The lanes 2A and 2B for strain M1B, lanes 3A and 3B for strain T1W , lanes 4A

and 4B for strain M3W (no insert), lanes 5A and 5B for strain T3W (no insert), lanes 6A

and 6B for strain M1W and lanes 7A and 7B for strain M2W. Lane 1, represents

molecular weight makers (hyperladder I) from Bioline.

Recombinant vectors from strains M1B, M1W, and M2W were purified using the Qiagen

Plasmid Midi Kit as described in materials and methods section 2.7.2, and then again

screened by PCR to check whether plasimds that harbour glpX were isolated or not. All

the purified plasmids gave positive results for inserts. Restriction digestions were

performed in order to reconfirm the orientation of the inserts. These purified plasmids

were used in the restriction digest reaction mixture as described in materials and

methods section 2.7.4. The size of the pCR2.1-TOPO vector is 3931 bp and PCR

fragment is 1111 bp, hence, the total size of the recombinant plasmid containing glpX

gene is 5042 bp.

1 A B A B A B A B A B A B 2 3 4 5 6 7

1Kbp_

10kbp_

0.6kbp_


80

The EcoRI enzyme has two restriction sites in the recombinant vector which can be

found at 11 bp upstream and at 6 bp downstream of the PCR product (Figure 3.7).

Therefore, after EcoRI digestion two fragments 1128 bp and 3914 bp should be formed.

In the case of pM1B, pM1W, and pM2W plasmids, EcoRI gave two products, one was

around 1000 bp which represents the insert and the second product was around 4000 bp

and that represents the vector (Figure 3.8). These results reconfirm that glpX was

successfully cloned in the plasmid which is consistent with the PCR screening results

However, HindIII restriction sites can be found 60 bp upstream from the insert and 302

bp from the 5`end of the PCR product (Figure 3.7). For the insert under control of the

T7 promoter, products of 869 bp and 4173 bp should be formed, however, products of

367 bp and 4675 bp should be formed for insert under the control of the lac promoter

(Figure 3.7). HindIII digestion of the plasmids pM1W and pM2W gave products of

around 800 bp and 4,000 bp (Figure 3.8), which indicates that the glpX gene is under the

control of the T7 promoter. Whereas for pM1B plasmid, HindIII produces two

products, one was around 5,000 bp and the second one around 400 bp which indicates

that the glpX gene is under the control of the lac promoter (Figure 3.8). These results

were consistent with the PCR screening.


81

Figure ‎3.7: Illustration of the strategy to determine the orientation of the insert in

the vector by restriction enzymes.

The blue circle represents the TOPO vector (3931 bp) and the white oblong represents

the cloned PCR product (1111 bp). (A) EcoRI cuts the TOPO vector 11 bp from the 5`

end and 6 bp from 3` end of the PCR product. (B) When the insert is under the control

of the lac promoter, HindIII cuts at 60 bp upstream of the 5` end and 307 bp

downstream from the 5` end of the PCR product. (C) when the insert is under the

control of T7 promoter, HindIII cuts at 60 bp upstream from the 5` end and 809 bp

downstream from 5` end of the PCR product.

PCR Product

EcoRI EcoRI

TOPO Vector

Digested fragment 1128 bp

11 bp 6 bp

(A)

PCR Product

HindIII HindIII

TOPO Vector


60 bp 307 bp

(B)

PCR ProductHindIII HindIII

TOPO Vector


60 bp 809 bp

(C)


82

Figure ‎3.8: Agarose gel showing HindIII (A) and EcoRI (B) digests of purified

plasmid.

In the case of plasmid pM1B which, HindIII produced two products in lane 2A; one was

around 5,000 bp and the second one around 400 bp. The HindIII digests of the plasmids

pM1W (lane 3A), and pM2W (lane 4A) gave products of around 800 bp and 4 kbp,

whereas, plasmid pM1B, pM1W, and pM2W, EcoRI digests produced two fragments,

one around 4 kbp and the other around 1 kbp in lanes B2, B3 and B4 respectively.

Lane 1, represents molecular weight makers (hyperladder I) from Bioline.

1kbp_

1 2 3 4A B A B A B

0.8kbp_

0.4kbp_

4kbp_

10kbp_

0.2kbp_


83

3.3.3 Growth of E. coli strains on different selective media for phenotype studies.

The phenotypes of all the E. coli mutant strains needed to be checked before

transferring the plasmids that harbour the glpX gene. LB Agar plates (+Tet) were used

to grow the E. coli strains JB103, JB108, JLD2402, JLD2403, and JLD2405. All these

strains are tetracycline resistant because they carry the Tn10 transposon (Donahue et al.,

2000). Also, the E. coli strains JB103, JB108 and JLD2402 all lack a functional fbp

gene and therefore cannot grow on gluconeogenic substrates such as glycerol. The

JB103 strain has multiple copies of the lac repressor gene which allows tight regulation

of the recombinant gene expression (Lutz & Bujard, 1997). Whereas, the JB108 strain,

which also lacks fbp, can express T7 RNA polymerase when induced with Isopropyl β-

D-1-thiogalactopyranoside (IPTG) (Donahue et al, 2000), and therefore, genes under the

control of the T7 promoter can be indirectly induced by the addition of IPTG to the

medium. Moreover, JB108 still has a functional glpX class II gene, but the expression

level of this gene is very low, hence, it is not sufficient for growth on glycerol (Donahue

et al., 2000). However, the JLD2402 strain is a double mutant (∆fbp and ∆glpX)

(Donahue et al., 2000). The strains were streaked onto M9 minimal medium that

contained 0.2% glucose or 0.4% glycerol (+Tet). All mutants grew on the M9 glucose

medium as expected. Whereas, only two out of five strains were able to grow on

glycerol, JLD2403 (fbp+, ∆glpX) and JLD2405 (glpX

+, fbp

+) (Figure 3.9), due to both

strains harbouring a functional fbp gene which enables them to grow on gluconeogenic

substrates such as glycerol (Table 3.4). The strains JB103, JB108 and JLD2402 were

unable to grow on glycerol due to the ∆fbp mutation (Table 3.4). However, all the E.

coli strains JB103, JB108, JLD2402, JLD2403, and JLD2405 were able to grow on LB

(Table 3.4). These results indicate that JB103, JB108 and JLD2402 can be used for

genetic complementation of the fbp mutation.


84

Table ‎3.4: Growth of the E.coli mutants on LB, glycerol and their phynotype.

Plus sign (+) represents growth and minus sign represents no growth.

Strain Genotype LB Glycerol

JB103 ∆fbp, glpX+ and lacIq + -

JB108 ∆fbp , glpX+

and T7 RNA polymerase + -

JLD2402 ∆fbp and ∆glpX + -

JLD2403 fbp+and ∆glpX + +

JLD2405 glpX+and fbp

+ + +

Figure ‎3.9: Growth of E. coli strains on M9 minimal medium containing 0.4%

glycerol.

Each section represents, 1: JB103, 2: JB108, 3: JLD2403, 4: JLD2402, 5: JLD2405, 6:

JB108.


85

3.3.4 Chemical transformation and complementation of different E. coli mutants.

Since the PCR analysis of plasmids pM1B and pM1W matched the restriction analysis in

term of insert orientation, the next stage was to transform selected E. coli mutant strains

with these plasmids that contain glpX gene. The aim of this step was to investigate

whether the C. acetobutylicum glpX gene does indeed encode for FBPase or not by

genetic complementation. Therefore, different E. coli ∆fbp mutants (JB103, JB108 and

JLD2402) were used to study various phenotypes exerted by the C. acetobutylicum glpX

gene and then to select the best transformant for enzyme assay. Purified plasmid pM1B

containing the insert under the control of lac promoter was transformed into E. coli

JB103 and JLD2402 mutants all of which lack a functional fbp gene. However, the

JB108 strain was transformed as described in section 2.6, using the pM1W plasmid

containing the insert under the control of T7 promoter because it can express T7 RNA

polymerase when induced with IPTG.

Due to the vector pCR2.1-TOPO harbouring a gene that encodes for ampicillin

resistantce, hence, transformants were spread out on LB plates containing ampicillin

and tetracycline (all the E. coli mutant strains are resistant to tetracycline) to confirm the

presence of plasmid in the transformants. After that, each colony was given a name

such as JB108/pM1W strain which indicates the host strain name and transformed

plasmid. Then colonies were transferred onto fresh LB plates (+Amp and +Tet), and to

M9 glycerol plates (+Amp and +Tet) to observe the differences in the phenotype and

whether the C. acetobutylicum glpX gene can complement the mutation or not.

The JB108/pM1W strain was also transferred onto M9 glycerol plates (+Amp and +Tet)

supplemented with IPTG to induce the expression of glpX. Surprisingly, the

JB108/pM1W strain showed no growth on the M9 glycerol medium containing IPTG

(Figure 3.10), whereas, in the absence of IPTG, a lot of colonies were formed. This

may be due to the cloned glpX gene harbouring an extra sequence upstream of the

transcriptional start site that RNA polymerase might recognise as a promoter.

Therefore, the cloned glpX gene would be expressed from that promoter rather than

from T7 promoter which means that IPTG is not needed (Table 3.5).


86

Another possibility is that addition of IPTG will potentially result in stimulation of

expression of the cloned DNA in both directions; and therefore a nonsense mRNA

expressed from the lac promoter might interfere with translation of the mRNA derived

from the T7 promoter.

Alternatively, the reason why the IPTG inhibits growth on glycerol may be that the

overproduction of the foreign clostridial GlpX protein is toxic to the E. coli JB108 strain

grow on glycerol minimal medium (a poor medium). Further investigations were

carried out by streaking JB108/pM1W on LB agar (+Amp and +Tet) supplemented with

IPTG (Figure 3.11) to test whether the effect of the overexpression of the GlpX protein

was still present in rich media. The growth on LB agar was not affected by the addition

of IPTG and this might indicate that cells can tolerate a high level of GlpX protein when

grown on rich medium such as LB (Figure 3.11 and Table 3.5)

Figure ‎3.10: Growth of JB108/pM1W strains on two M9 glycerol plates.

Plate (A) supplemented with IPTG (+Tet), and plate (B) without IPTG (+Tet). Control

is the mutant JB108 strain without the recombinant plasmid which harbours glpX.


87

Table ‎3.5: Growth of different E. coli mutant strains in LB and glycerol medium

supplemented with or without IPTG.

Plus sign (+) represents growth and minus sign represents no growth

Strains/plasmids LB LB (IPTG) Glycerol Glycerol (IPTG)

JLD2402/pM1B (lac) + Not tested + +

JB108/pM1W(T7) + + + -

JB103/pM1B (lac) + Not tested + Not tested

Figure ‎3.11: Growth of JB108/pM1W strains on two LB plates.

Plate (A) supplemented with IPTG (+Tet), and plate (B) without IPTG (+Tet). Control

is the JB108 strain without the recombinant plasmid which harbours glpX.


88

The JB103/pM1B and JLD2402/pM1B strains in which the cloned glpX gene is under

the control of the lac promoter, were tested on M9 glycerol medium (+Tet) together

with their controls, the JB103 and JLD2402 mutants. Only 15 colonies of JB103/pM1B

and only one colony of JLD2402/pM1B grew (Table 3.5 and Figure 3.12). Four out of

15 colonies of JB103/pM1B were transferred onto another M9 glycerol plate (+Tet) for

further confirmation (Figure 3.13). All the four colonies were able to thrive on the

glycerol plate. This could be due to that the JB103 strain contains a lac repressor gene,

which would lower the expression of the clostridial glpX gene that is under the control

of the lac promoter, thus reducing toxicity and this would allow the cells to thrive and

grow in poor media such as M9 glycerol. These results are consistent with the previous

finding that a high expression level of the clostridial glpX gene is toxic to the tested

JB108 mutant strain under glycerol growth conditions. However, poor growth was stil

observed after transferring the JLD2402/pM1B strain onto another M9 glycerol plate

(+Tet) (Table 3.5).


89

Figure ‎3.12: Growth of JB103/pM1W and JLD2402/pM1B on M9 glycerol plate.

Controls represent the JB103 and JLD2402 strain without the recombinant plasmid

which harbours glpX. Only one colony of the JLD2402 strain grew and 15 colonies of

JB103 grew on glycerol. The controls, JB103 and JLD2402, were unable to grow on

glycerol.

Figure ‎3.13: Growth of JB103/ M1B on M9 glycerol plate (+Tet).


90

3.3.5 Fructose bisphosphatase assay

Based on genetic complementation results, the JB108/pM1W strain was selected for

assay analysis due to the fact that the strain can tolerate overexpressed GlpX protein and

also was the only mutant available which harbours the T7 RNA polymerase to express

the glpX gene under the control of T7 promoter. A colony of the JB108/pM1W strain

was inoculated into 10 ml of LB broth (+Amp and +Tet) and incubated overnight at

37°C and then 5 ml was transferred into 500 ml of Overnight ExpressTM

Instant LB

medium and then incubated overnight at 37°C. The cells were broken using a French

Pressure Cell as described in the materials and methods section to produce a crude

extract and then the activity was assayed using by Beckman Coulter DU 800

spectrophotometer at 37°C. The protein concentration in crude extract (12.3 mg/ml)

was determined by the microbuiret assay, using bovine serum albumin as a standard.

The crude extract was used in a spectrophotometric coupled enzyme assay to measure

the FBPase activity by detecting the reduction of NADP+ to NADPH

spectrophotometrically at 37°C and wavelength of 340 nm. The activity of the coupling

enzymes, glucose-6-phophatase dehydrogenase and phosphoglucose isomerase were

confirmed using fructose 6-phosphate as substrate.

The assay was initiated by adding FBP to the 1 ml mixture. F-6-P will be formed as a

result of the FBPase reaction and this was converted to glucose 6-phosphate and then to

6-phosphogluconate by coupling to phosphoglucoisomerase and glucose-6-

dehydrogenase. As a result the NADP+ will be converted to NADPH which was

followed at 340 nm. All the reported classes of FBPase required divalent cations such

as Mg2+

, Mn2+

, Fe2+

, Cu2+

or Zn2+

to maximize the activity (Brown et al., 2009).

Therefore, all these divalent cations were tested. Maximum activity was obtained with

10 mM Mn2+

, whereas no activity was detected using Fe2+

, Cu2+

, Ca2+

or Zn2+

. No

activity was detected in the absence of a cation. Replacing Mn2+

with 10 mM Mg2+

reduced the activity about 75%. The effects of 1 mM of ATP, ADP, AMP, PEP, and

KH2PO4 on the FBPase activity were tested (Table 3.6). The optimal activity was

achieved using fructose 1,6-bisphosphate as substrate at pH 8.0. The FBPase activity of

GlpX was completely inhibited by the addition of 1 mM inorganic phosphate.

Moreover, 1 mM PEP reduced the activity by 16.5%, whereas, an increase in the

activity about 20.5% was detected after the addition of 1 mM ADP.


91

Other metabolites that were tested, AMP and ATP at 1 mM, produced no significant

effect on the FBPase activity of GlpX (Table 3.6). These results are comparable to

these obtained for GlpX proteins from M. tuberculosis and E. coli, except for the effect

of ADP and PEP. Although strain JB108 has a functional glpX gene in the chromosome,

it is not expressed at a sufficient level to enable grow on minimal medium

supplemented with a gluconeogenic substrate such as glycerol. Therefore, the glpX gene

in the chromosome is unable to compensate the loss of fbp gene (Donahue et al., 2000).

Crude extracts from JB108 mutant strain (control) were tested to detect any FBPase

activity of the chromosomal glpX in the extract and as expected no activity was

detected. Brown et al, 2009 reported that the E. coli GlpX enzyme is structurally

related to the Lithium-sensitive phosphatases which are strongly inhibited by lower than

0.3 mM Li+. Also they found that GlpX and YggF (another class II) were not sensitive

to lithium even at 10 mM Li+, hence, the class II FBPases in E. coli belong to the more

resistant Li+-sensitive phosphatases such as the class IV bifunction IMPase/FBPase

enzymes (Brown et al., 2009). This encouraged the testing of Li+ as a potential

inhibitor. No effect was detected after the addition of 1 mM or even 10 mM Li+, which

indicates that the C. acetobutylicum GlpX belongs to the more resistant Li+-sensitive

phosphatases. This assay again showed that glpX gene encodes for FBPase enzyme.

Table ‎3.6: The effect of different metabolites (effectors) on the reaction rate of the

crude GlpX enzyme compare to the control (no effector).

Effectors(1 mM) Reaction rate % (-)Inhibition or

(+)activation rate %

FBPase specific activity

(nmol/min/mg)

None 100 0 18.51 ± 0.88

ATP 93.7 -6.3 17.34 ± 0.04

ADP 120.5 +20.5 22.30 ± 0.79

AMP 93.1 -6.9 17.24 ± 0.11

PEP 83.5 -16.5 15.45 ± 0.13

Inorganic

phosphate

No activity No activity No activity


92

3.3.6 Analysis of sequenced recombinant plasmid

The recombinant plasmid from the JB108/pM1W strain that contains the insert under the

control of the T7 promoter was purified using a QIAGEN® plasmid midi kit. The region

that contains the insert was sequenced by Beckman Coulter Genomics using the T7

promoter and M13 reverse primers. No mutations or mismatches were detected in the

sequence analysis on comparision with the orginal putative C. acetobutylicum

chromosomal glpX gene.


93

3.4 Cloning fbp gene using TOPO cloning system.

Based on the BLAST search, the C. acetobutylicum fbp gene is an orthologue of the B.

subtilis fbp gene which belongs to the class III FBPase enzymes. The fbp gene was

amplified using the TOPOfbp primers (Table ‎2.5 and Table ‎3.7) and C. acetobutylicum

genomic DNA was used as a template with 49°C as annealing temperature. Two PCR

products were seen on an agarose gel, the first fragment was fbp with a size of around 2

kbp and the second fragment was an unwanted band of a size around 800 bp (Figure

3.14). Therefore, two different approaches were used to attempt to purify the fbp gene.

The first approach was using different annealing temperatures (48, 50 and 51°C) to try

produce a PCR product around 2 kbp without the unwanted fragment (Figure 3.14).

However, the unwanted fragment was still present even after using various annealing

temperatures. Therefore, to overcome the problem, the target fragment was purified

using a gel purification kit as described in materials and methods section 2.9.2. After

gel purification only the target gene of around 2 kbp was seen on agarose gel (Figure

3.14). The purified PCR product was cloned into the pCR2.1-TOPO vector and

transformed into E. coli Mach1™

-T1R and TOP10F` which as mentioned before has the

lacIq repressor to reduce the background expression of the cloned gene. After

transformation, PCR screening was carried out using the same primers, however, after

plating the transformants on LB agar supplemented with (+Amp and +Tet) and X-gal

for blue white screening , all the colonies were blue which indicated that these

transformants did not contain the fbp gene. Further confirmation was carried out by

PCR screening, however, no PCR product was detected on the agarose gel. These steps

were repeated several times with no success. After the fbp gene was cloned and

expressed using the Ligation Independent Cloning system (LIC) which will be

explained later on in the next chapter, attampts to clone it using the TOPO system were

discontinued.


94

Table ‎3.7: Locations of TOPOfbp primers on the sequence of cac1572 (fbp gene).

Bases underlined represent the location of TOPOfbp primers. Bases highlighted in

green and red represent the start codon and stop codon respectively. Obtained from

Genome Information Broker.


CCTTGAAAATGGCTATAAAGTTAAATTTGTTGACGGTAAAAGCATAATAATATAAAATACCATTAAGACA

CAAACGACACACTGTATACGGAGGGTTAAAATTATTATGCTATTAGAAAGTAACACCAAAAACGAAGAAA

TTAAGGACAATTTAAAGTACTTAGTTCTTCTTTCGAAACAGTACCCAACAATTAACGAAGCAGCTACGGA

AATAATCAATCTACAGGCTATTTTAAACCTACCTAAAGGAACGGAACACTTTTTATCAGATGTTCATGGG

GAATATGAACAATTCATACATGTACTTAAGAACGCCTCTGGAGTAATAAAAAGAAAAATAGACGATATTT

TCGGAAATAGACTTATGCAGAGTGAAAAGAAAAGTCTTGCTACGTTGATTTATTATCCGGAGCAGAAGCT

GGATATAATATTAAAGCAGGAAAAAAATATTGATGACTGGTATAAAATAACACTGTATAGGCTTATAGAG

GTTTGCAGGAATGTCTCCTCAAAGTATACTCGTTCTAAAGTAAGAAAAGCTCTTCCTAAAGAATTTTCGT

ATATAATTGAGGAGCTTTTGCATGAACAACCCAAGGGAGTAGATAAGCAGGAATATTATGACGAGATAAT

AAAGACTATTATAAGCATAGATAGGGCTAAGGAGTTTATAACTGCAATATCAAAGCTTATACAGAGGCTT

GTAGTAGATAGACTTCACATAATAGGTGATATCTTTGATAGAGGTCCAAGGGCGGATATTATAATGGATA

AGCTTGAAGAGTATCATGCGGTAGATATTCAATGGGGAAATCATGATATTTTATGGATGGGAGCTGCATC

CGGTTCTTCAGTATGCATGGCAAATGTAATAAGAATTTCTGCAAGATATGCAAATCTGTCAACTATAGAA

GATGGTTACGGAATTAATTTGTTGCCATTAGCCACTTTTGCTATGGACTTTTACGGAAACGATAAGTGCA

AGAATTTTGAACCTAAGATAGAATCTGATAAAAGTTATACAGTCAAGGAAATTGAACTTATAGGTAAAAT

GCATAAGGCTATTGCGATAATACAGTTTAAGCTTGAAGGAGAAGCTATAAAAAGACATCCTGAGTTTAAG

ATGGAACATAGGATGCTTCTAAACAAAATCAACTTTGAAGACAGTACCATAGAATTGGATGGTAAAAAGT

ATAAATTAAATGATACAAGCTTTCCAACTATAGATAAAAATGATCCATATAAACTTATAGATGAGGAGAG

GGAGGTAGTTGAAAAATTAAGATCTTCATTTGTAAATAGTGAGAAACTAAACAGGCATGTAAGGTTTTTA

TTTTCTCATGGAAATCTGTACCTAAAATTTAACTCTAATTTGTTATACCATGGCTGTATACCTTTAAATG

AGGACGGAACCTTTAAAGAGGTGTTAATAGGAAGTCATAAGTATAAGGGAAAGGCACTTTTAGATAAACT

TGATGTTTTAGCTAGAAAAAGCTTTTTTTATGAAGAAAATTCTAAAAACAGTAAGTATGAGAATGATATG

ATATGGTATCTTTGGTCAGGCCCTTTTTCGCCACTTTTTGGTAAAGAAAAAATGACAACATTTGAAAGAT

ATTTTATTGACGATAAGAAGACACATTATGAAAAGAAAGACCCATATTATCATTACAGAGATGATGAAGA

TATATGTATCAATATTTTAAGGGAATTTGGACTTGATTCTGAGCAAGCTCATATTATAAATGGGCATGTT

CCTGTAGAAAGTAAAAATGGAGAAAATCCAATAAAGGCAAATGGGAAATTAATAGTTATTGATGGAGGGT

TTTCAAAGGCATATCAAAGTAAAACTGGAATCGCTGGATATACCTTAATATATAACTCTTTTGGACTTCA

ACTTGTATCACATGAACTGTTTGAAACAACTGAAAAAGCAATAAAAGAAGAAACAGATATAATATCTTCT

TOPOfbp forward primer


95

ACTGTTATATTTGAAAAATCGGTAAAAGAAAAAGAGTAGGAGATACTGATATAGGGAAAGATTTGAAAAA

GCAGCTCTATGAGTTGAACCTATTGTTTTAGCTTATAAGAAGGGGCTTATTAAGGAATTTGTAAAAAGTT

AAGATTATGTACAAAAAAA

GAGAGATTTTATGTCCTCT

TOPOfbp reverse primer


96

Figure ‎3.14: Two agarose gels of PCR amplification and purification of fbp gene.

(A) amplification of C. acetobutylicum putative class III FBPase gene (fbp) without

purification using TOPOfbp primers forward and reverse and the unwanted fragment as

indicated by Lane 2. Lane 1, molecular weight makers (hyperladder I) from Bioline.

(B) Purified PCR product (fbp) indicated by lane 2. Lane 1, molecular weight makers

(hyperladder I) from Bioline.


97

3.5 Discussion

3.5.1 Cloning of glpX gene using TOPO cloning system

The bifunctional HPr kinase/phosphorylase is the most important element of carbon

catabolite repression in low GC Gram positive bacteria. It requires fructose 1,6-

bisphosphate for activation of kinase activity, however, inorganic phosphate is required

for activation of phosphorylase activity (Kravanja et al., 1999; Jault et al., 2000).

Genome sequence analysis and experimentation on C. acetobutylicum ATCC 824 have

revealed the presence of a HPr kinase/phosphorylase gene, hprK, which encodes a

protein of 304 amino acids and a molecular mass of around 34.5 kDa. Moreover, this

solventogenic organism shows a unique gene arrangement in which the HPr

kinase/phosphorylase gene (hprK) is followed by a gene encoding a putative class II

FBPase (glpX), hence, this gene might have a role in carbon catabolite repression

(Tangney et al., 2003). Until now, five classes of FBPase have been reported, and that

includes the class I, encoded by fbp which plays an essential role in the gluconeogensis

pathway and is found in almost all organisms (Sato et al., 2004). A class II enzyme was

first described in E. coli (Hines et al., 2006), whereas, class III was firstly reported in B.

subtilis (Fujta et al., 1998). The class V enzyme was found in archaea, and the class IV

enzyme was discovered in thermophilic prokaryotes of both domains (Brown et al.,

2009).

In E. coli, the class I FBPase is the main FBPase which is involved in gluconeogenesis.

Hence, a ∆fbp strain cannot grow on gluconeogenic substrates such as glycerol.

The glpFKX operon was found in E. coli and Shigella flexneri, encoding a glycerol

kinase (glpF), glycerol facilitator (glpK) and a gene of unknown function (glpX)

(Donahue et al., 2000; Truniger et al., 1992). The protein encoded by glpX was almost

identical in these two organisms (Truniger et al., 1992). Donahue et al, (2000) reported

that the GlpX protein had FBPase activity by overexpressing the glpX gene in a fbp

mutant to complement growth on glycerol. This protein has a molecular weight around

40 kDa and a dimeric structure (Donahue et al., 2000). Brown et al, (2009) reported

that another gene encodes for a FBPase class II enzyme (yggF). Therefore, there are

three genes (fbp, glpX and yggF) which encode for FBPase activity in E. coli; however,

the functions of glpX and yggF are still unkown (Donahue et al., 2000; Brown et al,

2009).


98

Several studies reported that M. tuberculosis can use fatty acids as a carbon source,

however, there was no gene assigned to encode for class I FBPase activity in the

genome sequence of M. tuberculosis. The Rv 1099c gene encodes a GlpX-like class II

FBPase that shares 43% identity to the E. coli class II FBPase (Movahedzadeh et al.,

2004). This gene complemented E. coli fbp mutants lacking FBPase activity indicating

that Rv 1099c is the main and only gene in M. tuberculosis that encodes for the missing

FBPase which is the key enzyme in the gluconeogenesis pathway (Movahedzadeh et al.,

2004). Also, another bacterium C. glutamicum, has only one main class II FBPase

enzyme, hence a fbp mutant cannot grow on gluconeogenic substrates (Rittmann et al,.

2003). The clostridial glpX gene encodes a protein showing 45% identity to the E. coli

class II GlpX and also has a molecular weight of 40 kDa. It was the aim of this study to

show that the clostridial GlpX protein has FBPase activity, which may be involved in

regulating the FBP levels in the cell, hence, regulating the important element in CCR

which is the bifunctional HPr kinase/phosphorylase activity and thus the phosphorylated

state of HPr on the serine-46 site.

E. coli mutant strains that lack the class I FBP were grown on Nutrient Agar and

minimal media to ensure the stable phenotype which was described by Donahue et al.,

(2000). As expected, all the strains matched the phenotype, being unable to grow on

glycerol due to lacking a functional class I fbp gene. It is important to test these

mutants on a gluconeogenic substrate, as a mutation that restored the growth on a

gluconeogenic substrate such as glycerol would give false results when cells are

transformed with the putative clostridial glpX gene.

This clostridial glpX gene was amplified from genomic DNA of the C. acetobutylicum

ATTC 824. Using a TOPO TA Cloning® Kit from Invitrogen, the PCR product was

ligated into the pCR2.1-TOPO vector and then transformed into E. coli Mach1™

-T1R

and TOP10F` cells. The transformation efficiency was poor, with a total of 14 white

colonies that may have the insert, many fewer than expected. However, this was a

sufficient number of clones to undertake the next stage of screening. The PCR

fragments can be inserted into the pCR2.1-TOPO vector in one of two orientations,

under the control of the T7 or lac promoter.


99

The desirable orientation is that the glpX gene would be placed in the pCR2.1-TOPO

vector under control of the lac promoter, as this would allow direct control of the glpX

expression in strains such as JLD2402 and JB103 that lacked both class I and II

FBPases. However, for the JB108 strain the glpX gene may be under the control of the

T7 promoter, due to this strain having T7 RNA polymerase which can be induced by

IPTG to express the glpX gene. Therefore, the JB108 strain was transformed with the

pCR2.1-TOPO vector containing the glpX gene under the control of the T7 promoter.

PCR screening was carried out to verify the orientation of the inserts using the

TOPOglpX forward and reverse primers along with the T7 promoter primer. Only one

PCR product should be produced, when the combination of TOPOglpX forward and T7

promoter primers was used for the inserts under the control of lac promoter. However,

when a combination of TOPOglpX reverse and T7 promoter primers was used, one

product which is under the T7 promoter would be given and no product for TOPOglpX

forward and T7 promoter primers. All the strains (T1W, M1W and M2W) produced

fragments from the combination of TOPOglpX reverse and T7 promoter primers

indicating that the insert was under the control of T7 promoter. Except one strain

termed M1B which produced a fragment from the combination of TOPOglpX forward

and T7 promoter primers, indicating that the insert was under the control of lac

promoter. This indicated that something was powerfully discriminating against an

insert under the control of the lac promoter. Surprisingly, the strain M1B produced blue

colonies when plated on a medium containing X-gal, even though an insert was present

in the pCR2.1-TOPO vector.

Restriction analysis of the recombinant plasmids from the strains M1B, M1W, and M2W

also indicated that the inserts in both M1W, and M2W were under the control of the T7

promoter. However, the glpX gene in strain M1B was under the control of the lac

promoter. Therefore, these results were consistent with PCR screening. Two fbp

mutant strains JB103 and JLD2402 were transformed with the M1B plasmid in which

the insert was under the control of lac promoter. Also the JB108 strain was transformed

with the M1W plasmid in which the insert was under the control of T7 promoter. The

main purpose of using different E. coli mutant strains to test genetic complemenation

was to investigate the phenotypes that were exereted by GlpX in order to find out what

is the best E. coli mutant that could be used in FBPase assay analysis.


100

All the JB103, JB108 and JLD2402 mutants were successfully transformed and able to

grow on LB (+Amp and +Tet). The mutant strain JLD2402 carries deletions in both the

fbp and glpX genes, which allows testing of the putative clostridial glpX FBPase

activity. Therefore, the transformed JLD2402 strain was streaked onto M9 glycerol

plates (+Tet). Only one colony grew on the plate, but this was enough to prove that

glpX encodes for an enzyme that has the FBPase activity. Also the JB103 strain which

contains a lac repressor allowing tight regulation of the insert under the control of the

lac promoter was tested on M9 glycerol plates (+Tet). Only 15 colonies were able to

grow on the glycerol plate but growth of these colonies showed that glycerol could be

used as a carbon source. The JB108 strain contained the T7 polymerase which can be

induced by IPTG to overexpress a cloned gene. Transformants of JB108 were tested on

two different glycerol plates, one with IPTG and the other one without IPTG. The

results were, no growth on the IPTG plate and a high level of growth on the plate

without IPTG, which indicates that the E. coli cells cannot cope with the over

expression of the foreign gene under poor growth conditions, which leads to inhibition

of growth.

Also, this strain was tested on LB plates one supplemented with IPTG and the other one

without IPTG. A high level of growth was seen on both plates, and a possible

explanation of this was that on a rich medium such as LB, the cells may tolerate the

high level of clostridial GlpX protein but, when transferred to poor environment such as

glycerol minimal medium, transformants cannot withstand the overexpression, of the

foreign protein and are hence unable to grow. The JB108/pM1W strain was selected for

FBPase assay analysis due to the fact that the GlpX protein can be overexpressed by

Overnight Express TM

Instant LB media with inhibiting the growth.

To assay the activity, the strain JB108/pM1W was grown on Overnight Express TM

Instant LB medium, to make crude extracts for testing the FBPase activity. The enzyme

activity was demonstrated using a coupled enzyme assay technique which linked the

production of fructose 6-phosphate to the reduction of NADP+ to NADPH (Jules et al.,

2009; Brown, et al., 2009; Donahue et al.,2000). The addition of cell extracts to the

reaction mixture resulted in formation of NADPH which can be detected by an increase

of the absorbance at 340 nm. Crude cell extract from the mutant JB108 strain was also

made to be tested as a control, and as expected no activity was detected for this mutant.


101

Although as mentioned before, this strain does contain the glpX gene, the low level of

expression of this gene cannot support growth on gluconeogenic conditions and enzyme

activity cannot be detected by the coupled enzyme reaction (Donahue et al., 2000).

The GlpX activity was optimized using Mn2+

as other reported FBPase enzymes such

as class I, II, and III need divalent cations as such Mg2+

, Zn2+

, and Mn2+

for maximium

activity (Brown et al., 2009; Donahue et al.,2000 ). Also other divalent cations

including Fe2+

, Cu2+

, Ca2+

and Ni2+

were tested but no activity was detected. The effects

of AMP, ADP, ATP, and PEP (1 mM) on GlpX activity were tested. ATP and AMP (1

mM) had almost no effect on the activity of C. acetobutylicum GlpX enzyme (Table

3.8), whereas, in E. coli, the GlpX enzyme was not effected by AMP and ADP (1 mM).

Moreover, ATP inhibited the activity only by 12%, but inorganic phosphate inhibited

the E. coli GlpX activity completely (Table 3.8) (Brown, et al., 2009; Donahue et al.,

2000). On the other hand, ATP and AMP (1 mM) had no effect on the C. glutamicum

GlpX enzyme (Rittmann et al., 2003). Moreover, inorganic phosphate inhibited the

activity completely, suggesting that phosphate may have a regulatory role on GlpX

activity in C. acetobutylicum ATCC 824 (Table 3.8).

PEP (1 mM) stimulated the GlpX activity in E. coli by 1.7-fold, however, no effect was

detected in M. tuberculosis GlpX (Table 3.8). But in B. subtilis, PEP (1 mM) inhibited

the GlpX activity completely (Jules et al., 2009). In this study, however, PEP inhibited

the GlpX activity only by 16.5%. However, ADP (1 mM) does not exert any effect on

GlpX activity for any of the bacteria reported (Table 3.8). Instead, in this study, GlpX

activity was stimulated by 20.5% (Table 3.8). The complete inhibition via inorganic

phosphate was explained by solving the 3D structure of E. coli in which inorganic

phosphate was shown to block the active site of the enzyme (Brown, et al., 2009). This

indicates that same mechanism might be responsible for complete inhibition by

inorganic phosphate in C. acetobutylicum. Also, C. acetobutylicum GlpX was shown to

belong to resistant Li+-sensitive phosphatases such as E. coli GlpX due to the fact it was

not completely inhibited by even 10 mM Li+. Unlike the M. tuberculosis GlpX which is

inhibited by by more than 90% at 2.5 mM.


102

Table ‎3.8: Comparision of the effect of various metabolites on the activity of different GlpX enzymes.

microorganism Effectors (inhibit or stimulate FBPase activity of GlpX ) Source

ATP ADP AMP PEP Phosphate

E. coli Inhibited by

12% (1 mM)

No effect

(1 mM)

No effect

(1 mM)

Stimulate by 1.7-fold

(1mM)

Inhibited completely

(1 mM)

Brown et al., 2009;


M. tuberculosis Not tested No effect

(1 mM)

No effect

(1 mM)

No effect (1 mM) Not tested Movahedzadeh et al.,

2004

C. glutamicum No effect

(1 mM)

No effect

(1 mM)

Inhibited

completely

(90 µM)

Inhibited only by 50%

(360 µM)

Inhibited only by 50%

(1.6 mM)

Rittmann et al., 2003

C. acetobutylicum Inhibited by

6.3% (1 mM)

Stimulated

by 20.5%

(1 mM)

Inhibited by

6.9% (1 mM)

Inhibited by 16.5%

(1 mM)

Inhibited completely

(1 mM)

This study

B. subtilis Not tested Not tested Not tested Inhibited completely

(1 mM)

Not tested Jules et al., 2009


103

3.5.2 Cloning of fbp gene using TOPO cloning system

The putative fbp gene in C. acetobutylicum encodes for a class III Fbp enzyme. To date,

this class has only reported in B. subtilis (Fujita et al., 1998; Jules et al., 2009). Based

on bioinformatic analysis the size of in C. acetobutylicum enzyme was 77.23 kDa which

is almost twice the size of the GlpX class II enzyme (34.81 kDa). Therefore, this

enzyme needs to be investigated as a second potential FBPase protein in C.

acetobutylicum. The assay results of B. subtilis class III FBPase enzyme indicate that

PEP is needed for full activity and adding PEP (1 mM) to the assay reaction mix

resulted in 30-fold stimulation compared to a control with no effector added to the assay

reaction mix (Jules et al., 2009). Also in B. subtilis, this gene enables the cell to grow

on a gluconeogenic substrate such as glycerol even after disruption of the glpX gene

which might imply that the fbp gene is the main FBPase enzyme in B. subtilis that is

involved in the gluconeogensis pathway. However, recent results revealed that glpX

gene can substitute for the FBPase after fbp has been knocked out (Jules et al., 2009).

Significant problems were encountered in cloning the C. acetobutylicum fbp gene using

the TOPO cloning system. After amplification of fbp, two PCR products were detected,

the first fragment was the target which was around 2 kbp and the second fragment was

small unwanted band of around 800 bp. It was attempted to solve this problem by two

different techniques, the first was changing the annealing temperature from 49 to 48, 50

and 51°C with no sucess. Secondly, the amplified fbp was purified using gel

purification kit to ensure that only the target fragment which was fbp gene would be

cloned into the vector. After the transformation, no clones were detected that had the

fbp gene which might indicate that the fbp gene was toxic to the E. coli host strain

which would prevent the growth of the transformed cells. Problems in cloning genes

encoding an FBPase enzyme have been reported by Col, (2004). He could not clone the

yggF which is the third gene in E. coli that encodes FBPase enzyme, but he had no

explanations for this problem. However, this gene was cloned later (after 5 years) by

Brown et al, (2009) using an expression vector obtained from the Genobase collection.


104

3.6 Conclusion

In this chapter, the open reading frame cac1088 was investigated using bioinformatic

analysis which showed that this open reading frame might encode for a class II FBPase

enzyme (GlpX). The putative glpX gene was cloned using TOPO cloning technology,

and transformed into an E. coli fbp mutant that cannot grow on glycerol. As a result,

the transformed cells were able to grow on glycerol which indicates that glpX does

indeed encode for FBPase activity. Different phenotypes were detected by growing the

transformants which carry glpX on glycerol and LB agar with or without IPTG. The

JB108/pM1W strain could grow on LB agar with or without IPTG, but no growth was

detected when the JB108/pM1W strain was streaked on glycerol agar supplemented with

IPTG. Instead, cells were able to grow on glycerol without IPTG. This might imply

that overpression of the C. acetobutylicum glpX gene is toxic to the E. coli mutant

(JB108) on this medium. FBPase activity was detected by assaying crude extract of the

JB108/pM1W strain in the presence of 10 mM Mn2+

. The GlpX enzyme was

completely inhibited by 1 mM inorganic phosphate and stimulated about 20.5% by 1

mM ADP. To date, this is the first study to report FBPase activity in clostridia.

Attempts to clone the putative fbp gene, which might encode for Fbp class III were

unsuccessful using TOPO cloning technology.


105

Chapter 4:

4 GlpX and Fbp overexpression and

purification


106

4.1 Gateway cloning system

4.1.1 Introduction

To assay FBPase activity of purified GlpX and Fbp enzymes, cloning, overexpression

of both proteins and then purification needed to be carried out. Different

overexpreesion strategies were explored in order to obtain the possible highest

expression and purity level without aggregation. The first expression system that was

explored was the Gateway expression system (Invitrogen®). The Gateway

® Technology

is based on two infectious stages of the bacteriophage lambda in E. coli. Bacteriophage

lambda can enter the lysogenic pathway, in which it integrates itself into the genomic

DNA of E. coli at a specific site or alternatively it releases active progeny viruses in a

process called lytic pathway. In the lysogenic pathway, the integrated phage can either

be excised from the host genome and go back to lytic behavior or can be transferred to

daughter cells. There are four att binding sites, termed attP, attB, attL, and attR

respectively, that govern the integration and excision process of the phage lambda DNA

catalysed by integrase (Int) and excisionase (Xis) enzymes encoded by the phage

lambda genome, and the E.coli integration host factor (IHF). The integration reaction,

called the BP reaction (attB×attP) and the excision reaction, called the LR reaction

(attL×attR). The gene of interest can be integrated or excised in vitro by the BP or LR

reactions, and this is the foundation of the Gateway®

cloning Technology.

Attempts were made to express the fbp and glpX of C. acetobutylicum genes by the

Gateway system, two reactions were applied by creating an entry clone for each gene

with the Donor vector pDONR™

221 (BP reaction) and then transferring each gene to

the expression vector pET-60-DEST (LR reaction). PCR primers were designed to

amplify each gene flanked by two attB overhangs (attB1 and attB2) to enable the

recombination with two attP regions (attP1 and attP2) in the donor vector

pDONR™221 (BP reaction) to form an entry clone with two attL sites (attL1 and

attL2) (Figure 4.1).

After the integration reaction, the entry clone was mixed in the LR reaction with the

expression vector (pET-60-DEST) (Appendix) that contains attR sites (attR1 and

attR2), so that each gene was transferred from the entry clone (pDONR™

221)

(Appendix) to the expression vector (pET-60-DEST).


107

Figure ‎4.1: Illustration of the two BP and LR reactions of Gateway® cloning

Technology.

(A) BP Reaction: Facilitates recombination of an attB substrate (attB-PCR product)

with an attP substrate (donor vector) to create attL sites in the entry clone and this

reaction is catalyzed by BP ClonaseTM

II enzyme mix. (B) LR reaction: Facilitates

recombination of an attL substrate (entry vector) to create attB sites in the expression

clone and this reaction is catalyzed by LR ClonaseTM

II enzyme mix. Figure taken from

Invitrogen Gateway manual, 2009.

(A)

(B)


108

4.1.3 Results

4.1.3.1 Cloning of the glpX gene‎into‎the‎donor‎vector‎pDONR™221

To clone the glpX gene into the donor vector pDONR™

221 of the Gateway system, the

GATEglpX primers (Table ‎2.5 and Table ‎4.1) were designed to amplify the glpX gene

with the attB overhangs, with C. acetobutalicum genomic DNA used as template. The

amplified PCR product consisted of the 975 bp glpX gene, flanked by a 31 bp attB site

at the 5` terminus, and a 30 bp attB site at the 3` terminus.

In the integration reaction (BP reaction), the two attB sites of the PCR products

recognize the two attP sites in the pDONR™

221 vector and are recombined by integrase

enzyme to form the entry clone, in which the glpX gene was integrated into

pDONR™

221 vector. The reaction mix was transformed into One Shot® OmniMAX

™

2-T1R chemically competent E. coli and then plated on LB (+Kan) for screening

(Section ‎2.8.2). Cells that contain plasmid without the integrated glpX gene could not

grow due to the presence of ccdB gene (Figure ‎4.1) which is lethal to E. coli by

inhibiting gyrase which has the ability to relax the negative supercoiling DNA to allow

replication (Bernard, 1996). Therefore, makes the screening for transformants that

contain the glpX gene straightforward. Twelve Colonies out of over 300 resistant to

(+Kan) were selected, and then PCR screening (Section ‎2.7) was carried out with the

same primers that were used to amplify the glpX gene. A product of around 1 kbp was

observed (Figure ‎4.2). Thus, indicated that the plasmid containing glpX has

successfully transformed into E. coli. The plasmids from three different selected

isolates MAX-glpX-1, MAX- glpX-8, and MAX glpX-10 were purified (Figure ‎4.3) as

described section ‎2.7.2. These purified plasmids gave different sizes on the gel even

though their size around 6 kbp due to the fact that the plasmid can exist in two forms;

supercoiled and relaxed (Figure 4.3).


109

Table ‎4.1: Locations of GATEglpX primers on the sequence of cac1088 (glpX

gene).

Bases underlined represent the locations of GATEglpX primers. Bases highlighted in




ATCAAGGAGGGAAATTCATGTTTGATAATGATATATCCATGAGTTTAGTAAGAGTAACAGAAGCAGCAGC

ACTACAATCTTCAAAGTATATGGGAAGAGGAGATAAAATTGGAGCTGATCAAGCAGCAGTAGATGGAATG

GAGAAGGCATTTAGTTTTATGCCAGTAAGAGGCCAGGTTGTAATAGGAGAGGGAGAACTTGATGAAGCTC

CTATGCTTTATATAGGTCAAAAGCTTGGTATGGGAAAAGACTATATGCCTGAAATGGATATAGCAGTAGA

TCCTTTAGATGGAACGATTTTAATTTCTAAGGGACTACCTAATGCAATAGCAGTAATAGCAATGGGACCA

AAAGGAAGTTTACTTCATGCCCCAGATATGTATATGAAGAAAATTGTTGTGGGACCTGGAGCAAAAGGTG

CTATAGATATAAATAAATCTCCTGAAGAGAATATTTTAAATGTAGCAAAGGCATTAAACAAGGACATATC

TGAATTAACAGTTATAGTTCAAGAAAGAGAAAGACATGACTACATAGTAAAAGCAGCTATAGAAGTTGGA

GCAAGAGTTAAGCTATTTGGTGAGGGCGATGTTGCAGCTGCACTTGCTTGTGGTTTTGAAGATACTGGAA

TAGACATACTTATGGGAATTGGAGGAGCTCCAGAAGGAGTTATAGCCGCAGCAGCTATCAAGTGCATGGG

CGGAGAAATGCAGGCTCAGCTTATACCTCATACTCAGGAAGAAATAGATAGATGTCACAAAATGGGAATA

GATGATGTAAATAAAATTTTCATGATAGATGATTTAGTTAAAAGTGATAATGTGTTTTTTGCAGCTACAG

CAATAACAGAATGTGATCTTCTTAAGGGCATAGTATTTTCTAAAAATGAACGTGCAAAAACCCATTCCAT

ATTAATGAGATCT

AAAACTGGTACAATAAGATTTGTTGAAGCTATTCATGACTTGAATAAAAGTAAATTAGTGGTAGAATAAA

GATEglpX forward primer

GATEglpX reverse primer



110

Figure ‎4.2: Agarose gel of PCR screening of the glpX gene in MAX-glpX isolates.

Twelve different isolates (MAX-glpX) were used for PCR screening as indicated by

lane 2 to 13. Lane 1 represents molecular weight makers (hyperladder I) from Bioline.

Figure ‎4.3: Agarose gel of the purified plasmids (pMAX-glpX).

Three plasmids (recombinant pDONR™

221 carrying glpX) were purified from different

colonies. Lane 2, 3, and 4 represent pMAX-glpX-1, pMAX-glpX-8, and pMAX-glpX-

10 plasimds respectively. Lane 1, represents molecular weight makers (hyperladder I)

from Bioline.

1Kbp_

1 2 3 4 5 6 7 8 9 10 11 12 13

10kbp_

0.2kbp_

1 2 3 4

10kbp_

0.4kbp_

6kbp_


111

4.1.3.2 Transferring the glpX gene into the expression vector pET-60-DEST

The glpX gene was transferred from the donor vector to the expression vector pET-60-

DEST by the LR reaction (excision reaction), in which the attL sites at each end of the

glpX gene in pDONR™

221 and the attR sites in the expression vector pET-60-DEST

were recombined. Thus, the lethal ccdB gene was replaced in the pET-60-DEST vector

by the glpX gene (Figure ‎4.1). The reaction mixture was transformed into the

expression host, E. coli Rosetta™

2(DE3), and transformants were selected on LB plated

agar (+Amp). Twelve colonies out of over 300 were screened by PCR using the same

cloning primers. The PCR products from 12 (termed Rosetta-glpX-1-12) different

isolates were around 1 kbp as expected (Figure 4.4).

Figure ‎4.4: Agarose gel of PCR screening of the glpX gene in Rosetta-glpX isolates.

Twelve Rosetta-glpX isolates were used for PCR screening as indicated by lane 2 to 13.


1 2 3 4 5 6 7 8 9 10 11 12 13

1Kbp_

10kbp_

0.4kbp_


112

Two colonies (Rosetta-glpX-6 and Rosetta-glpX-7) were selected for plasmid

purification, and carried out as described in material and methods section ‎2.7.2. After

purification, the two plasmids pRosetta-glpX-6 and pRosetta-glpX-7 (recombinants

pET-60-DEST carrying glpX) were examined via sequencing (Section ‎2.7.3) with

GATEglpX primers by Beckman Coulter Genomics. The sequencing result was a

disappointment, with a lot of mismatching and unidentified bases, even although Easy-

A High-Fidelity PCR Cloning Enzyme had been used to amplify glpX for the cloning in

the donor vector. The same problem was found also with other students using this

system to express different genes.


113

4.1.3.3 Cloning of the fbp gene into the donor vector pDONR™

221

To clone the fbp gene into the donor vector pDONR™

221 of the Gateway system, the

GATEfbp primers were designed to amplify the fbp gene with attB overhangs (Table

‎2.5 and Table ‎4.2), and the C. acetobutylicum genomic DNA was used as template. The

amplified PCR products consisted of the 2000 bp fbp gene, flanked by a 31 bp attB site

at the 5` terminus, and a 30 bp attB site at the 3` terminus.

The fbp gene was cloned in the same way as glpX (Section ‎0). After transformation of

the recombinant pDONR™

221 into One Shot® OmniMAX

™ 2-T1R chemically

competent E. coli, transformants were plated on LB agar (+Kan) for screening. Four

colonies out of over 250 that were resistant to (+Kan) were isolated for potential

insertion and then PCR screening was carried out with the same primers that used in

clone fbp gene and the product of this pair was the same size as that one used in the

cloning, around 2 kbp (Figure 4.5). This Thus indicated the plasmid that contains fbp

has successfully transformed into One Shot® OmniMAX

™ 2-T1R Chemically

Competent E. coli. The plasmids from two different clones MAX-fbp-1 and MAX-fbp-

2 were purified as described in section ‎2.7.2 and their size was around 9 kbp (Figure

‎4.6). After purification, the two plasmids (pMAX-fbp-1, pMAX-fbp-2) were examined

via sequencing (Section‎2.7.3) with same cloning primers (GATEfbp primers) by

Beckman Coulter Genomics. Again the sequencing result was a disappointment, with a

lot of mismatching and unidentified bases.


114

Table ‎4.2: Locations of GATEfbp primers on the sequence of cac1572 (fbp gene).

Bases underlined represent the locations of GATEfbp primers. Bases highlighted in
































GCAGCTCTATGAGTTGAACCTATTGT

TTTAGCTTATAAGAAGGGGCTTATTAAGGAATTTGTAAAAAGTTAAGATTATGTACAAAAAAA

GATEfbp forward primer

GATEfbp reverse primer



115

Figure ‎4.5: Agarose gel of PCR screening of the fbp gene in MAX- fbp isolates.

Three isolates (MAX-fbp-1, 2, 3 and 4) were used for PCR screening as indicated by

lanes 2, 3, 4 and 5. Lane 1, represents molecular weight makers (hyperladder I) from

Bioline.

Figure ‎4.6: Agarose gel of the purified plasmids (pMAX-fbp).

Two plasmids (recombinant pDONR™

221 carrying fbp) were purified from different

colonies (pMAX-fbp-1, pMAX-fbp-2) as indicated by lanes 2 and 3 respectively.


1 2 3 4 5

2Kbp_

10kbp_

0.4kbp_

1 2 3 4 5

10kbp_

8kbp_


116

4.2 Cloning and expression of the glpX and fbp genes using Ligation Independent

Cloning system (LIC)

4.2.1 Introduction

To overcome the problem of unsatisfying cloning in the Gateway clonig system, another

overexpression system was investigated which is the Ligation Independent Cloning

system (LIC) from Novagen. This system was developed for directional cloning of

amplified PCR products with no need for restriction enzymes, DNA ligase or alkaline

phosphatase (Aslanidis and de Jong 1990). The system uses compatible 15 base single

stranded overhangs, which are created in the amplified PCR product by the 3'

5'

exonuclease activity of T4 DNA polymerase and anneal to the same complementary 15

base single stranded overhangs in the vector (Figure ‎4.7).

Figure ‎4.7: Diagram of the Ek/LIC strategy.

After amplification with primers that include the indicated 5' LIC extensions, the PCR

insert is treated with LIC-qualified T4 DNA Polymerase (+dATP), annealed to the

Ek/LIC vector, and the resultant nicked, circular plasmid is transformed into competent

E. coli. Figure taken from Novagen® LIC manual, 2010.


117

To create PCR products with the required overhangs, 15 extra bases is added to the

primers at the 5` end. The PCR products after the purification are treated by T4 DNA

polymerase in the presence of dATP to create specific vector-compatible overhangs and

then the treated PCR product is annealed to the pET-41 Ek/LIC vector. To express glpX

and fbp genes by the LIC system, the recombinant plasmid was transformed into the

expression host BL21 (DE3) pLysS.

4.2.2 Results

4.2.2.1 Amplification and purification of the glpX and fbp PCR products

To clone the glpX and fbp genes into the pET-41 Ek/LIC vector of the LIC system, the

LICglpX primers (Table ‎4.3 and Table ‎2.5) were designed to amplify the glpX gene and

also the LICfbp primers (Table 4.4 and Table ‎2.5) were designed (Section 2.1.2) to

amplify the fbp gene with the extra 15 bases overhangs at each terminus, using the C.

acetobutylicum genomic DNA as template. For the glpX gene, the amplified PCR

products consist of the 975 bp of the cloning sequence, flanked by the 15 extra bases at

each terminus. For the fbp gene, the amplified PCR products consist of the 1998 bp of

the cloning sequence, flanked by the 15 extra bases at each terminus (Figure ‎4.8).

Figure ‎4.8: Agarose gel of PCR amplification of the glpX and fbp genes.

Lanes 2 and 3, represent amplified fbp and lanes 4 and 5, represent amplified glpX.


1 2 3 4 5

2Kbp_

1Kbp_

10kbp_

0.6kbp_


118

Table ‎4.3: Locations of LICglpX primers on the sequence of cac1088 (glpX gene).

Bases underlined represent the locations of LICglpX primers. Bases highlighted in




ATCAAGGAGGGAAATTCATGTTTGATAATGATATATCCATGAGTTTAGTAAGAGTAACAGAAGCAGCAGC

ACTACAATCTTCAAAGTATATGGGAAGAGGAGATAAAATTGGAGCTGATCAAGCAGCAGTAGATGGAATG

GAGAAGGCATTTAGTTTTATGCCAGTAAGAGGCCAGGTTGTAATAGGAGAGGGAGAACTTGATGAAGCTC

CTATGCTTTATATAGGTCAAAAGCTTGGTATGGGAAAAGACTATATGCCTGAAATGGATATAGCAGTAGA

TCCTTTAGATGGAACGATTTTAATTTCTAAGGGACTACCTAATGCAATAGCAGTAATAGCAATGGGACCA

AAAGGAAGTTTACTTCATGCCCCAGATATGTATATGAAGAAAATTGTTGTGGGACCTGGAGCAAAAGGTG

CTATAGATATAAATAAATCTCCTGAAGAGAATATTTTAAATGTAGCAAAGGCATTAAACAAGGACATATC

TGAATTAACAGTTATAGTTCAAGAAAGAGAAAGACATGACTACATAGTAAAAGCAGCTATAGAAGTTGGA

GCAAGAGTTAAGCTATTTGGTGAGGGCGATGTTGCAGCTGCACTTGCTTGTGGTTTTGAAGATACTGGAA

TAGACATACTTATGGGAATTGGAGGAGCTCCAGAAGGAGTTATAGCCGCAGCAGCTATCAAGTGCATGGG

CGGAGAAATGCAGGCTCAGCTTATACCTCATACTCAGGAAGAAATAGATAGATGTCACAAAATGGGAATA

GATGATGTAAATAAAATTTTCATGATAGATGATTTAGTTAAAAGTGATAATGTGTTTTTTGCAGCTACAG

CAATAACAGAATGTGATCTTCTTAAGGGCATAGTATTTTCTAAAAATGAACGTGCAAAAACCCATTCCAT

ATTAATGAGATCT

AAAACTGGTACAATAAGATTTGTTGAAGCTATTCATGACTTGAATAAAAGTAAATTAGTGGTAGAATAAA

A

LICglpX forward primer

LICglpX reverse primer



119

Table ‎4.4: Locations of LICfbp primers on the sequence of cac1572 (fbp gene).

Bases underlined represent the locations of LICfbp primers. Bases highlighted in green

and red represent the start codon and stop codon respectively. Obtained from Genome

Information Broker.






























GCAGCTCTATGAGTTGAACCTATTGT

TTTAGCTTATAAGAAGGGGCTTATTAAGGAATTTGTAAAAAGTTAAGATTATGTACAAAAAAA

LICfbp forward primer

LICfbp reverse primer



120

In order to clone the glpX and fbp genes in the LIC system a large amount of ultra pure

PCR products was needed. Therefore, PCR amplification was carried out again to allow

DNA purification from an agarose gel (Section 2.3). After amplification, 60 µl of PCR

products of both genes instead of 5 µl was loaded onto the agarose gel for DNA agarose

gel purification procedure and purified as described in materials and methods section

2.9.2. This step is to ensure that only the band of interest is purified (Figure 4.9).

Figure ‎4.9: Agarose gel of purified PCR products of the glpX and fbp genes using

agarose gel purification (Ultrafree-DA).

Lanes 2 and 3, represent glpX and lane 4, fbp. Lane 1, represents molecular weight

makers (hyperladder I) from Bioline.

1 2 3 4

1Kbp_

2Kbp_

10kbp_

0.4kbp_


121

Another purification step is also needed to remove dNTPs and residual enzyme and this

was done by chloroform extraction and ethanol precipitation method. The samples from

the agarose gel purification were extracted with chloroform and DNA was precipitated

with isopropanol as described in section 2.9.2 and again run in duplicate on an agarose

gel to show purity (Figure 4.10).

Figure ‎4.10: Agarose gel of purified PCR products of the glpX and fbp genes

following chloroform extraction and ethanol precipitation.

Lanes 2 and 3, represent glpX and lanes 4 and 5, represent fbp. Lane 1, represents



122

4.2.2.2 Cloning the glpX into the pET-41 Ek/LIC vector

After purification of the PCR products, T4 DNA polymerase treatment was carried out

to create compatible overhangs on the purified insert as described in section 2.9.3. The

treated insert was used in the annealing reaction with the pET-41 Ek/LIC vector. The

recombinant vector that contained glpX insert was transformed into NovaBlue

GigaSingles™

Competent Cells (cloning host). The transformants (Nova-glpX) were

plated on LB (+Kan). Seven colonies out more than 200 were selected for PCR

screening using the same primers that were used in cloning glpX. All the selected

screened colonies contained the plasmid pET-41 Ek/LIC vector that harbour the glpX

insert (Figure 4.11).

Figure ‎4.11: Agarose gel of PCR screening of the glpX gene in Nova-glpX isolates.

Seven isolates (Nova-glpX-1 to Nova-glpX-7) were PCR screened as indicated by lanes

2 to 8. Lane 1, represents molecular weight makers (hyperladder I) from Bioline.

Two (Nova-glpX-3 and Nova-glpX-4) from the seven isolates that contain the insert

were used for plasmid purification. The recombinant pET-41 Ek/LIC vector was

purified as described in section 2.7.2 and their sizes were around 7 kbp (Figure 4.12).

The two purified plasmids, pNova-glpX-3 and pNova-glpX-4, were again screened by

PCR using the cloning primers, and as expected both plasmids contained the glpX gene

(Figure 4.13).


123

Figure ‎4.12: Agarose gel of the purified pNova-glpX plasmids.

Two plasmids (pNova-glpX-3 and pNova-glpX-4) were purified from two different

colonies (Nova-glpX-3 and Nova-glpX-4) as indicated by lanes 2 and 3. Lane 1,


1 2 310kbp_

0.2kbp_

6kbp_


124

Figure ‎4.13: Agarose gel of PCR screening of the glpX after purification from

Nova- glpX-3 and Nova- glpX-4 isolates.

Two purified plasmid (pNova-glpX-3 and pNova-glpX-4) were PCR screening as

indicated by Lanes 2 and 3. Lane 1, represents molecular weight makers (hyperladder I)

from Bioline.

The two purified plasmids pET-41 Ek/LIC containing the glpX gene (pNova-glpX-3 and

pNova-glpX-4) were examined via sequencing with same cloning primers. The

sequencing results indicate that the glpX gene was cloned into the pET-41 Ek/LIC

vector.

4.2.2.3 Expression of the glpX gene in the recombinant pET-41 Ek/LIC vector

The purified plasmid pNova-glpX-3 was selected to be transformed into the expression

host, BL21 (DE3) pLysS. This strain contains an IPTG-inducible T7 RNA polymerase,

and is designed for protein expression from a pET vector. pLysS means that the strain

carries a pACYC184-derived plasmid that encodes T7 lysozyme, which is an inhibitor

of T7 RNA polymerase, and hence, represses basal expression of the target gene under

the control of the T7 promoter to reduce possible toxicity. Therefore, the plasmid

pNova-glpX-3 was transformed into the BL21 (DE3) pLysS cells as described in section

2.9.5. The transformants (BL21/pNova-glpX-3) were plated on LB (+Kan). To

demonstrate that the pNova-glpX-3 we successfully transformed into the expression

host, PCR screening (Section 2.9.6) was carried out for 4 selected isolates out of more

than 200 using the same cloning primers (Figure 4.14).

1 2 3

1Kbp_

10kbp_

0.2kbp_


125

Figure ‎4.14: Agarose gel of PCR sreening of the glpX gene in BL21/ pNova-glpX-3

isolates.

Four different isolates (BL211/pNova-glpX-3 to BL214/pNova-glpX-3) were PCR

screened. Lanes 2 to 5, represent amplified glpX. Lane 1, represents molecular weight

makers (hyperladder I) from Bioline.

To express the glpX gene, 500 ml of Overnight ExpressTM

Instant LB medium (Section

2.9.7) was used to grow the transformant (BL211/pNova-glpX-3), and this was

incubated overnight at 37°C. The cells were harvested and a crude extract prepared

using a French Pressure Cell Press®

as described in section ‎2.7.6. The recombinant

GlpX protein was purified under native conditions from the crude extract using the Bug

Buster® Ni-NTA His•Bind Purification kit as described in section ‎2.9.8, and then all the

purified fractions collected were analyzed by SDS-PAGE method as described in

section ‎2.9.9 (Figure ‎4.15). The recombinant fusion GlpX was purified to homogeneity

as estimated by SDS-PAGE with a molecular weight of about 73 kDa (Figure ‎4.15)

which was as expected and is bigger than the native protein which is 34.81 kDa, due to

the extra two His-Tags, GST-Tag, and S-Tag (appendix 7.1).

1Kbp_

1 2 3 4 5 6

10kbp_

0.4kbp_


126

These tags are important for the expressed recombinant protein, since they increase the

solubility, the efficiency of purification and prevent aggregation of the recombinant

protein. This was demonstrated by analysising the insoluble fraction of the GlpX crude

extract togather with the 2X diluted insoluble fraction of the GlpX crude extract in

SDS-PAGE gel in order to identify any aggregation, however, no sign of aggregation

was detected (Figure ‎4.15). The total molecular weight of all the tags in the

recombinant fusion protein is about 37 kDa. Thus, the total expected molecular weight

of the recombinant fusion protein is 37 + 34.81 = 71.81 kDa, which is very close to size

of the GlpX (about 73 kDa) seen on the SDS-PAGE gel (Figure ‎4.15).

Figure ‎4.15: SDS-PAGE of purified GlpX proteins in the eluted fractions after His-

tag purification under native conditions.

Lanes 1 and 2, insoluble fraction of the crude extract and 2X diluted insoluble fraction

of the GlpX crude extract respectively . Lanes 5 to 8, eluate fractions of GlpX. Lane 4,

HyperPage prestained protein marker (Bioline).


127

4.2.2.4 Cleavage of the recombinant fusion GlpX protein

Before assay of the recombinant GlpX enzyme, attempts were made to cut the

recombinant fusion tag part of GlpX using enterokinase. This enzyme is able to remove

all fused GST-Tag, His-Tag, and S-tag in the N-terminal from the recombinant fusion

GlpX by cleaving at the C-terminal end of the lysine residue which is before the start

codon site of the target gene (Figure 4.16).

Figure ‎4.16: Illustration of GlpX protein attached to tagged proteins at the N- and

C-terminus. Enterokinase cleavage site is shown by the horizontal arrow.

During purification, the recombinant GlpX protein was eluated using 1 M imidazole.

Since the enterokinase enzyme is inhibited by imidazole, the recombinant protein was

dialyzed against distilled water using a Dialysis cassette Slide-A-Lyzer as described in

materials and methods section 2.9.10, and then the dialyzed recombinant GlpX was

added to the reaction mix containing the enterokinase (Section 2.9.11). The

enterokinase enzyme appeared however to be unable to cut the tag region from the

recombinant GlpX, despite various optimizations of the reaction mix such as increasing

the amount of the recombinant protein from 10 to 20 μl, and changing the incubation

temperature (room temperature to 37°C). This might have been due to the inhibition of

some remaining imidazole in the reaction mix.


128

4.2.2.5 Assay of the fructose 1,6 biphosphatase activity of the purified

recombinant GlpX protein.

The purified recombinant GlpX was dialyzed against 50 mM Tris-Cl (pH8) containing 1

mM dithiothreitol (DTT) and 5 mM MgCl2. The recombinant GlpX was assayed as

described before in materials and methods section 2.7.6. However, no activity was

detected, a similar problem has also reported by Movahedzadeh et al, (2004) in that the

purification of recombinant GlpX from M. tuberculosis led to the loss of FBPase

activity.

4.2.2.6 Overexpression of the glpX gene in the mutant JB108

As an alternative means of demonstrating FBPase activity associated with the GlpX

enzyme, the purified plasmid pNova-glpX-3 containing the glpX gene was transformed

into the expression host, the mutant E. coli JB108 strain which is the same strain used

for genetic complementation in chapter 3. The transformants (JB108/ pNova-glpX-3)

were plated on LB (+Kan and +Tet) and more than 200 colonies were able to grow on

the plate. To demonstrate that the recombinant plasmid was successfully transformed,

two colonies (JB1081/pNova-glpX-3 and JB1082/pNova-glpX-3) were PCR screened

using the same cloning primers and both produced a PCR product of around 1 kbp

(Figure 4.17).

Figure ‎4.17: Agarose gel of PCR screening of the glpX gene in the JB108/ pNova-

glpX-3 isolates.

Two different isolates JB1081/pNova-glpX-3 and JB1082/pNova-glpX-3 were PCR

screened as indicated by lanes 2 and 3. Lane 1, represents molecular weight makers


1Kbp_

1 2 310kbp_

0.2kbp_

http://mic.sgmjournals.org/search?author1=F.+Movahedzadeh&sortspec=date&submit=Submit


129

The same overexpression procedure that was used with BL21 (DE3) pLysS was applied

to overexpress the GlpX protein from JB1081/pNova-glpX-3 strain to demonstrate

whether overexpression was possible in the JB108 mutant. The recombinant GlpX

protein was purified under native condition from the crude extract by Bug Buster® Ni-

NTA His•Bind Purification kit and then all the purified fractions were run in the SDS-

PAGE (Figure 4.18). As expected, all the purified proteins were around 73 kDa which

was consistent with the previous purification.

Figure ‎4.18: SDS-PAGE of purified GlpX proteins in the eluted fractions after His-

tag purification under native conditions.

Lanes 2 to 5, eluate fractions of GlpX. Lane 1, represents the HyperPage prestained

protein marker (Bioline).


130

4.2.2.7 Complementation of JB108 mutant

Before assaying the enzyme, the JB1081/pNova-glpX-3 and JB1082/pNova-glpX-3 need

to be tested via genetic complementation to know whether the recombinant fusion GlpX

still has FBPase activity or not. Therefore, two colonies of JB1081/pNova-glpX-3 and

JB1082/pNova-glpX-3 grown on LB plate (+Kan and +Tet) which harbour plasmid

pNova-glpX-3 were streaked on M9 glycerol plate (+Tet). As expected no growth was

detected due to the glpX gene being under the control of the T7 promoter, and the JB108

strain does not express the T7 RNA polymerase without the presence of IPTG in the

medium (Figure 4.19). Therefore, 50 µl of IPTG (200 mg/ml) was spread onto a M9

glycerol plate (+Tet), and the same two colonies were streaked onto it and then

incubated overnight at 37°C. The two colonies were able to grow on the medium, and

this indicates that the cloned glpX gene is expressed and has an FBPase activity (Figure

4.19).

Figure ‎4.19: Growth of JB1081/pNova-glpX-3 and JB1082/pNova-glpX-3 strain on

two M9 glycerol plates.

Plate (A) M9 glycerol medium (+Tet) without IPTG and plate (B) M9 glycerol medium

(+Tet) supplemented with IPTG. The JB1081/pNova-glpX-3 and JB1082/pNova-glpX-3

that were tested are indicated by 1 and 2 respectively.


131

4.2.2.8 Assay the fructose 1,6-biphosphatase activity of the recombinant GlpX

from JB1081/pNova-glpX-3 crude extract.

The genetic complementation results shown that the recombinant fusion GlpX had

FBPase activity. This indicates that the recombinant fusion tag part did not affect the

activity of GlpX enzyme. This encouraged the testing of the FBPase activity of

recombinant GlpX. Therefore, the JB1081/pNova-glpX-3 strain was grown on the same

overexpression media that used to grow the BL21 (DE3) pLysS strain and a crude

extract was prepared as described in section ‎2.7.6. The protein concentration of the

crude extract was 23 mg/ml and the FBPase activity was assayed using fructose 1,6-

bisphosphate as substrate at pH 8.0 (Section ‎2.7.6). The recombinant GlpX from

JB1081/pNova-glpX-3 strain had FBPase specific activity of 29.70 ± 0.61 nmol/min/mg.

Different divalent cations were tested (Mn2+

, Ca2+

, Zn2+

, Ni2+

, Cu2+

and Mg2+

), and 10

mM of Mn2+

was found to support the activity of GlpX as also shown in section ‎3.3.5.

No activity was detected when Mn2+

was omitted from the reaction mix. However,

replacing Mn2+

with 10 mM of Mg2+

led to increase the activity about 2-fold compared

to the 10 mM of Mn2+

. In contrast, no FBPase activity was detected when Mn2+

was

replaced with 10 mM of Zn2+

or Ca2+

. Moreover, all divalent cations that inhibit the

FBPase activity (Ca2+

, Zn2+

, Ni2+

, Cu2+

) were tested as a potential inhabitor for the

coupling enzymes that are used in the assay reaction. Ca2+

and Zn2+

did not inhibit the

activity of the coupling enzymes, whereas, Ni2+

, Cu2+

in inhibited the activity of these

enzymes completely.

The FBPase activity of GlpX was completely inhibited by the addition of 1 mM

inorganic phosphate. The PEP (1 mM) reduced the activity by about 26.5%. Other

metabolites (effectors) tested such as AMP, ADP and ATP, each at 1 mM each,

produced no major effect on the FBPase activity of GlpX (Table ‎4.5). These results are

compatible to that obtained for GlpX protiens from M. tuberculosis, except for PEP

(Movahedzadeh et al., 2004). Also, Li+ was tested as a potential inhibitor. No effect

was detected after the addition of 1 mM Li+, on the other hand, only 21% inhibition was

detected after the addition of 10 mM Li+, which reconfirms that the C. acetobutylicum

GlpX belongs to the more resistant Li+-sensitive phosphatases.


132

Table ‎4.5: The effect of different metabolites (effectors) on the reaction rate of the

crude recombinant GlpX enzyme compare to the control (no effector).

Effector (1mM) Reaction rate (%) Inhibition rate (%) FBPase specific activity

(nmol/min/mg)

Control 100 0 29.70 ± 0.61

ATP 93.7 6.3 28.83 ± 0.68

ADP 100 0 29.70 ± 0.08

AMP 100 0 29.70 ± 0.06

PEP 73.5 26.5 21.74 ± 0.49

Inorganic

phosphate



133

4.2.2.9 Cloning and expression of the fbp gene

After the second purification of the PCR product (Section 4.2.2.1), T4 DNA polymerase

treatment was carried out to create compatible overhangs on the purified insert as

described in section 2.9.3. The treated insert was used in the annealing reaction, by

adding the treated insert that contained the compatible overhangs to the pET-41 Ek/LIC

vector. The recombinant vector that contains fbp insert was transformed into NovaBlue

GigaSingles™

Competent E.coli Cells (cloning host) and the transformants were plated

on LB (+Kan). Five different colonies (designated from Nova-fbp-1 to Nova-fbp-5)

were PCR screened (Section 2.9.6) using the same primers that were used in cloning

fbp. All the selected screened colonies contained the insert (Figure 4.20). Two isolates

(Nova-fbp-3 and Nova-fbp-4) from the five were used for plasmid purification. The

recombinant pET-41 Ek/LIC vector was purified as described in section 2.7.2 and gave

a band size of around 8 kbp (Figure 4.21). The two purified plasmid (pNova-fbp-3 and

pNova-fbp-4) were PCR screened using the same cloning primers and as expected both

plasmid contain fbp gene (Figure 4.22). These two purified plasmids were examined

via sequencing with same cloning primers, and the sequencing results indicated that the

fbp gene was perfectly cloned into the pET-41 Ek/LIC vector.


134

Figure ‎4.20: Agarose gel of PCR screening of the fbp gene in Nova-fbp isolates.

Five isolates (Nova-fbp) were PCR screened as indicated by lane 2 to 6. Lane 1,


Figure ‎4.21: Agarose gel of the purified pNova-fbp plasmids.

Two plasmids (pNova-fbp-3 and pNova-fbp-4) were purified from two different

colonies as indicated by lanes 2 and 3. Lane 1, represents molecular weight makers


1 2 3 4 5 6 7 8 9 10 11 12 13

1 2 3 4 5 6 7 8

10Kb-

1Kb-

1 2 3

10kbp_

0.2kbp_

8kbp_


135

Figure ‎4.22: Agarose gel of PCR screeing of the fbp gene after purification.

Two purified plasmid (pNova-fbp-3 and pNova-fbp-4) were PCR screened as indicated

by lanes 2 and 3 respectively. Lane 1, represents molecular weight makers (hyperladder

I) from Bioline.

1 2 3

2Kbp_

10Kbp_

0.2Kbp_


136

4.2.2.10 Expression of the fbp gene in the recombinant pET-41 Ek/LIC vector

The purified plasmid pNova-fbp-3 was selected to be transformed into an expression

host, BL21 (DE3) pLysS. The transformants (BL21/pNova-fbp-3) were plated on LB

(+Kan). To show that the recombinant plasmid was successfully transformed, PCR

screening (Section 2.9.6) was carried out using the same cloning primers and a 2 kbp

band was observed on the agarose gel (Figure 4.23).

Figure ‎4.23: Agarose gel of PCR screening of the fbp gene in BL21/pNova-fbp-3

isolates.

Three different isolates (BL211/pNova-fbp-3, BL212/pNova-fbp-3 and BL213/pNova-

fbp-3) were used for PCR screening as indicated by lane 2 to 4. Lane 1 represents


The fbp gene was expressed in BL211/pNova-fbp-3 and purified in the same way as

glpX gene (Section 4.2.2.3). The recombinant fusion Fbp was purified to homogeneity

and estimated by SDS-PAGE to have a molecular weight of about 115 kDa (Figure

4.24), bigger than the native protein which is 77.23 kDa, due to the extra His-Tag, GST-

Tag, and S-Tag. The total molecular weight of all the tags in the recombinant fusion

protein is about 37 kDa. Thus, the total expected molecular weight of the recombinant

fusion protein is 37 + 77.23 = 114.23 kDa, which is very close to the Fbp size seen on

the SDS-PAGE 115 kDa (Figure 4.24). Also, no aggregation was detected in the

insoluble fraction of the Fbp crude extract together with the 2X diluted insoluble

fraction of the Fbp crude (Figure 4.24).

1 2 3 4 5 6 7 8 9 10 11 12 13

1 2 3 4 5 6 7 8

1 2 3

10Kb-

1Kb-

1 2 3 4 5 6 7 8

2Kbp_

0.2kbp_

10kbp_


137

Attempts to cut the recombinant fusion tag from the recombinant Fbp using

enterokinase enzyme was carried out (Section 2.9.11). As expected, the fusion tag in

the C-terminal was not removed. Also, an attempt was made to assay the purified

recombinant Fbp enzyme, however, no activity was detected.

Figure ‎4.24: SDS-PAGE of the purified Fbp proteins in the eluate fractions after

His-tag purification under native conditions.

Lanes 1 and 2, insoluble fractions of crude extract and the 2X diluted insoluble fractions

recepectively. The lane 3, empty and lane 4, HyperPage prestained protein marker

(Bioline). Lane 5 to 8, eluate fractions of Fbp.


138

4.2.2.11 Expression of the fbp gene in the mutant JB108

The problem of unable to detect FBPase activity was solved by transforming, the

purified plasmid pNova-fbp-3 into the E.coli JB108 mutant strain, the transformants

(JB108/pNova-fbp-3) were plated on LB supplemented with (+Kan and +Tet). To

confirm that the purified plasmid was transformed into the JB108 mutant strain. PCR

screening (Section 2.9.6) was carried out using the same cloning primers (Figure 4.25).

The fbp gene was shown to be expressed in JB1083/pNova-fbp-3 and purified in the

same way as the glpX gene had been (Section 4.2.2.6) and then all the purified fractions

were run in the SDS-PAGE to check whether Fbp was expressed or not (Figure 4.26).

Figure ‎4.25: Agarose gel of PCR screening of the fbp gene in JB1083/pNova-fbp-3

and JB1084/pNova-fbp-3 isolates.

Two different isolates (JB1083/pNova-fbp-3 and JB1084/pNova-fbp-3) were used for

PCR screening as indicated by lane 2 and 3, fbp fragments. Lane 1, represents


1 2 3 4 5 6 7 8 9 10 11 12 13

10Kb-

1Kb-

1 2 3 4 5 6 7 8

2Kbp_

10kbp_

0.2Kbp_


139

Figure ‎4.26: SDS-PAGE of the purified Fbp proteins in the eluate fractions after

His-tag purification under native conditions.

Lanes 2 to 5, eluate fractions of Fbp. Lane 1 represents the HyperPage prestained

protein marker (Bioline).

1 2 3 4 5

78KDa_

120KDa_

178KDa_


140

4.2.2.12 Complementation of JB108 mutant via fbp gene

The transformants (JB1083/pNova-fbp-3 and JB1084/pNova-fbp-3) cells were plated on

LB plate (+Tet and +Kan). Then the two colonies were transformed onto M9 glycerol

plate (+Tet). As expected no growth was detected in the palte on M9 glycerol plate

(+Tet) without IPTG (Figure 4.27). However, the transformants (JB1083/pNova-fbp-3

and JB1084/pNova-fbp-3) were able to on the M9 glycerol plate (+Tet) supplemented

with IPTG, this implies that the cloned fbp gene has FBPase activity (Figure 4.27).

Figure ‎4.27: Growth of JB1083/pNova-fbp-3 and JB1084/pNova-fbp-3 strains on

two M9 glycerol plates.

Plate (A) without IPTG (+Tet) and plate (B) with IPTG (+Tet). The JB1083/pNova-fbp-

3 and JB1084/pNova-fbp-3 that were tested are indicated by 3 and 4 respectively.


141

4.2.2.13 Assay of the fructose 1,6 biphosphatase activity of the recombinant Fbp

protein from a crude extract of the JB1083/ pNova-fbp-3

The protein concentration of the crude extract of JB1083/pNova-fbp-3 was17.2 mg/ml

as assessed by the Biuret methond (Section ‎2.7.6.1) and FBPase activity in was assayed

as mentioned in section ‎2.7.6. The recombinant Fbp from JB1083/pNova-fbp-3 strain

had FBPase specific activity of 747.67 ± 20.97 nmol/min/mg. Different divalent cations

were tested as metioned before (Mn2+

, Ca2+

, Zn2+

, and Mg2+

). The divalent cations used

that support the optimal activity of Fbp was Mn2+

(10 Mm) (Table ‎4.6). No activity was

detected when Mn2+

was absent from the reaction mix. However, replacing Mn2+

with

10 Mm of Mg2+

or Ca2+

reduced the activity about 59% and 93% respectively, compares

to the 10 Mm of Mn2+

. Instead, no FBPase activity was detected when Mn2+

was

replaced with 10 mM of Zn2+

.

The FBPase activity of Fbp was completely inhibited by the addition of 1 mM inorganic

phosphate. However, 1 mM of PEP and ATP reduced the activity about 10%, whereas,

1 mM of AMP reduce the activity about 16%. Also 1 mM of ADP was tested which

produced no major effect on the FBPase activity of Fbp (Table ‎4.6). As C.

acetobutylicum GlpX belongs to the more resistant Li+-sensitive phosphatases it was not

completely inhibited by 1 mM or 10 mM Li+

as mentioned before (Section 4.2.2.8) ,

hence, Li+ was tested as a potential inhibitor. Only 27% reduction in the activity was

detected after the addition of 1 mM Li+, however, a 45% reduction in activity was

detected after the addition of 10 mM Li+, which indicates that the C. acetobutylicum

Fbp also belongs to the more resistant Li+-sensitive phosphatases.


142

Table ‎4.6: The effect of different metabolites on the reaction rate of the recombinant

Fbp enzyme compare to the control (no effector).

Effector (1mM) Reaction rate (%) Inhibition rate (%) FBPase specific activity

(nmol/ min/mg)

None 100 0 747.67 ± 20.97

ATP 90.6 9.4 677.33 ± 10.56

ADP 95.3 4.7 712.21 ± 3.00

AMP 84.4 15.6 630.81 ± 34.12

PEP 90.5 9.5 680.23 ± 16.4

Inorganic

phosphate



143

4.2.2.14 Comparision of the FBPase activities of recombinant GlpX (native and

tagged) and recombinant Fbp proteins

The recombinant fusion GlpX was more active (29.70 ± 0.61 nmol/min/mg) by 1.6 fold

compared to the native GlpX (18.51 ± 0.88 nmol/min/mg) due to the recombinant

fusion GlpX being more concentrated than the native GlpX based on SDS-PAGE.

However, the recombinant fusion Fbp had activity (747.67 ± 20.97 nmol/min/mg)

which was 25 fold higher than the recombinant fusion GlpX and around 40 fold higher

than the native GlpX even though the concentration of the recombinant fusion GlpX

was higher than Fbp based on SDS-PAGE.

On the other hand, ADP had almost no effect on the activity of the recombinant fusion

GlpX, in fact, ADP stimulated the activity of the native GlpX by 20.5%. Also, the PEP

inhibited the activity for both proteins, with more inhibition 10% for the recombinant

fusion GlpX (Table 4.7). Phosphate inhibited the activity completely of both enzymes

which indicates that the active site is not affected by the presence of the N-terminal tags

in these recombinant fusion proteins (Table 4.7). In contrast, the ADP had no dramatic

effect on the FBPase activity of the recombinant fusion Fbp protein, whereas, the AMP

inhibited the activity by about 15.6%, however, only 9.4% and 9.5% inhibition were

detect after adding the ATP and PEP respectively (Table 4.7). Moreover, ATP excerted

similar inhibition rate on both native and recombinant GlpX enzymes.

Table ‎4.7: Comparisons of different effectors on the FBPase activity of GlpX

(native and tagged) and Fbp tagged of C. acetobutylicum.

Effectors (1mM) Percentage of Activity of FBPase enzymes compare to their control

(no effector)

GlpX (native) GlpX (tagged) Fbp (tagged)

ATP 6.3% inhibition 6.3% inhibition 9.4% inhibition

ADP 20.5% stimulation no effect 4.7% inhibition

AMP 6.9% inhibition no effect 15.6% inhibition

PEP 16.5% inhibition 26.5% inhibition 9.5% inhibition

Phosphate Complete inhibition Complete inhibition Complete inhibition


144

The recombinant fusion GlpX was stimulated 2-fold when the Mn2+

was replaced by

Mg2+

compared to about 75% inhibition for the native GlpX which might indicate that

the GST•Tag, and S•Tag have more drastic effect on the activity toward divalent

cations. In contrast, Ni2+

, Cu2+

, Zn2+

, Ca2+

were tested as potential divalent cations, no

activity were detected (Table 4.8). Also, the Ni2+

, Cu2+

, Zn2+

, Ca2+

were test as

potential inhibitors of the coupling enzymes in the assay reaction. Ni2+

and Cu2+

were

found to inhibit the coupling enzymes, indicating that the Ni2+

, Cu2+

most not used as

potential divalent cations alongside with coupling enzymes in assay FBPase activity.

Table ‎4.8: Comparisons of different divalent cations require for FBPase activity

of GlpX (native and tagged) and Fbp tagged of C. acetobutylicum.

Divalent cations

(10 mM)

Percentage of Activity of FBPase enzymes compare to control

condition (10 mM of Mn

2+)

GlpX (native) GlpX (tagged) Fbp (tagged)

Mg2+

75% inhibition 200% stimulation 60% inhibition

Ni2+

Inhibit the coupling

enzymes


enzymes


enzymes

Cu2+

Inhabit the

coupling enzymes

Inhabit the coupling

enzymes


enzymes

Zn2+


Ca2+

No activity No activity Only 6% activity

When the Mn2+

was replaced by Mg2+

, about 60% loss of the activity of the recombinant

fusion Fbp was detected, compare to 2 fold increase in the activity of the recombinant

fusion GlpX and about 75% inhibition for the native GlpX. This might indicate that

attached tags affect the metal bind site (Table 4.8).

Moreover, a small activity 6% compare to control was detected when Mn2+

was

replaced by Ca2+

, to date this is the first study reported that Fbp class III has shown

activity with Ca2+

as divalent cations that required for FBPases enzyme activity (Table

4.8). On the other hand, the Zn2+

, Ca2+

were test as potential divalent cations, no

activity were detected.


145

These results shown that the N-terminal tags had only a very small effect on the activity

of the target protein (GlpX) probably due to the fact that the tagged proteins was

engineered on the surface of the target protein and away from the active site to reduce

any effect in the activity of the target protein. These small differences in the behaviour

toward the effectors compare to the native GlpX due to the GST•Tag, and S•Tag have

the ability to enhance the folding of the target protein which may contribute to very

small increase or decrease on the activity in the presence of effectors.


146

4.3 Discussion

4.3.1 Overexpression of GlpX and Fbp proteins by Gateway cloning system.

This expression system was used in order to purify the recombinant GlpX and Fbp

proteins and then assay these pure proteins. The Gateway technology is based on the

specific-site recombination reactions catalyzed by the bacteriophage λ Integrase (Int)

protein (Hartley et al., 2000). The Gateway cloning system consists of two

bacteriophage pathways, the BP reaction is the first step that mediates by the

bacteriophage λ Integrase (Int) and the E. coli Integration Host Factor (IHF) proteins

and this mix of enzymes is termed BP ClonaseTM

II enzyme mix and this step called

lysogenic pathway. The second step of this system is the LR reaction which is the lytic

pathway catalyzed by the LR ClonaseTM II enzyme mix that consists of the

bacteriophage λ Integrase (Int), Excisionase (Xis), and E. coli Integration Host Factor

(IHF) proteins. The BP ClonaseTM

II enzyme mix and LR ClonaseTM

II enzyme mix

recognise specific-sites (att-sites). The GATEglpX and GATEfbp primers are designed

to amplify glpX and fbp genes flanked with 31 bp attB sequences at both ends. These

PCR products are mixed together with the Donor vector (pDONR™221) which contains

the attP sites, and BP ClonaseTM

II enzyme mix in the first step of cloning reaction (BP

reaction). As a result, the entry clone was formed containing the gene of interest and by

product. The entry clone was transformed into One Shot® OmniMAX™ 2-T1R

Chemically Competent E. coli.

Transformed cells grew on the LB plates (+Kan) which indicated that an entry clone

had successfully transformed. The transformed cells that contain the Donor vector

(pDONR™

221) without the cloned genes cannot grow due to the ccdB which a lethal

gene which located at the cloning site. Therefore only the transformed cells that

harbour the entry clones with glpX or fbp genes grew. Moreover, PCR screening

reconfirmed these results by amplifying the glpX or fbp genes from the entry clones.

These entry clones containing glpX or fbp genes were purified and the second step

which is the lytic pathway (LR reaction) was conducted with the LR ClonaseTM

II

enzyme mix and the destination vector harbours the lethal gene (ccdB) that surrounded

by attR sequences.


147

The expression clones were formed containing the glpX or fbp genes and by product

vector which contains the lethal gene. These mixtures were transformed in Rosetta™

2(DE3) Competent Cells (expression strain). Meanwhile, the purified entry clones

containing the glpX or fbp genes were sent for sequencing and the results were

disappointments due to a lot of mismatching and unidentified bases as mentioned

before. Although the glpX and fbp genes were amplified using the Easy-A high

fidelity PCR cloning enzyme which has a proofreading activity (3´- to 5´-exonuclease

activity) that removes or excises the wrong base pair and replaced with a correct one

(Newton and Graham, 1994). Moreover, this enzyme has a six fold lower error rate

compared to Taq DNA polymerase and two to three fold lower than any proofreading

archaeal DNA polymerases in the market (Strategene®

manual, 2009). Therefore, it is

unlikely to have errors or mismatching in the PCR products. Also, the same problem

was seen with another student using different genes and due to these problems, we

discontinued the use of the Gateway cloning system and switched to Ligation

Independent Cloning system.

4.3.2 Overexpression of GlpX and Fbp proteins by LIC system

This expression system does not require T4 ligase or restriction enzymes, however, in

order to clone a gene, primers were designed with an extra 15 bp at 5` ends. The

amplified PCR product as a result, would include the gene of interest flanked with the

extra 15 bp at the ends. However, this system is based on the 3'

5' exonuclease

activity of T4 DNA Polymerase together with dATP which removes all the extra 13 bp

(dGTP, dCTP, and dTTP) from 3'

5' direction and stop at the 13 bp which is

adenosine. Therefore, the 5` compatible overhangs on the PCR product is generated,

which anneals to the compatible overhangs on the the Ek/LIC vector. The glpX and fbp

genes were amplified using LICglpX and LICfbp primers respectively which contain

compatible overhangs at 5` ends for easy cloning with no the needs of T4 ligase. Two

PCR products were detected on the agarose gel, a fragment about 1 kbp of the amplified

glpX and the other fragment was round 2 kbp for amplifying fbp. Moreover, the LIC

system is sensitive to any nonspecific amplified fragments, remaining dNTPs and DNA

polymerase from PCR and these reduce the efficiency of the cloning reaction.


148

Therefore, two purification procedures were used, the first one was the agarose gel

purification to remove any unwanted PCR products and this step avoids any possibility

of nonspecific cloning. The second step was Chloroform: Isoamyl Alcohol (CIAA)

extraction and isopropanol precipitation to inactivate DNA polymerase and remove the

remaining dNTPs which prevented interference with T4 DNA Polymerase. After

purification, the glpX and fbp amplified PCR products were treated with T4 DNA

Polymerase and dATP to create compatible overhangs at 5` ends and cloned into pET-

41 Ek/LIC plasmid.

In this plasmid, the protein encoded by the cloned gene is fused with N-terminal

GST•Tag, His•Tag, S•Tag and also another His•Tag in C-terminal, these tags are to

facilitate the purification and solubilization of recombinant protein. The Glutathione S-

transferase (GST•Tag™) is a 211 amino acid protein with a molecular mass of 26 kDa

which makes it the largest fused protein. Moreover, the GST•Tag is often integrated

with other tags such as His•Tag to increase the solubility of the recombinant protein and

prevent aggregation. However, some recombinant proteins may aggregate even though

they contain a His•Tag fusion and this is why pET-41 Ek/LIC plasmid which harbours

more than one tagged fusion protein was selected and also, to increase the purity of the

target protein. However, the disadvantage of using a large tag is that the fused protein

has to be removed for some applications such as crystallization (Terpe, 2003).

Meanwhile, the polyhistidine tag (His•Tag) is the smallest tag consisting of six histidine

amino acids attached to the N-terminal of the recombinant protein and another one is

attached to the C-terminal (Appendix 7.7). The advantage of using small tags is that

some applications such as crystallization can be conducted without interference (Brown

et al., 2009; Terpe, 2003).

After cloning, the plasmids that harboured the fbp and glpX genes were transformed into

NovaBlue GigaSingles™ Competent E.coli Cells. These were excellent cloning hosts

with high transformation efficiency and this step was used to increase the yield of the

plasmids containing fbp and glpX genes. PCR screening was carried out using the same

primers that have been used before in the amplification of fbp and glpX genes. As

expected, Two PCR products were detected on the agarose gel, a fragment about 1 kbp

of the amplified glpX and the other fragment was round 2 kbp for amplifying fbp.


149

The plasmids were purified and then transformed into an expression host, BL21 (DE3)

pLysS, a strain genetically engineered for expression vectors such as pET vectors. Also

this strain contains the T7 bacteriophage gene 1(DE3), encoding T7 RNA polymerase

and harbours the pLysS plasmid, which carries the gene encoding T7 lysozyme that

lowers the background expression of the target gene under the control of T7 promoter

under uninduced conditions. However, T7 lysozyme, under induced conditions, which

was achieved by adding IPTG, does not interfere or lower with the expression level of

the target gene (Davanloo et al., 1984). This creates an excellent environment to

increase the yield of target protein without killing the cells. After transformation to the

expression host, PCR screening was conducted to confirm the presence of the plasmids

that harbour the fbp or glpX genes. The cells were induced for glpX and fbp expression

and the GlpX and Fbp proteins were purified using the Bug Buster® Ni-NTA His•Bind

Purification kit.

As mentioned before, the expressed Fbp and GlpX were fused with two hexahistidine

tags at N and C terminals. Therefore, after loading the crude extract into the column,

covalent bonds were formed between the two His•Tags on the surface of Fbp and GlpX

proteins and Ni, whereas, the rest of the proteins that not did have His•Tags were among

the flow through. To elute the proteins, imidazole was added which competes with and

replaces the tagged Fbp and GlpX proteins in attaching to the immobilized metal ion.

As a result, the fused Fbp and GlpX proteins were purified to homogeneity estimated by

SDS-PAGE, moreover, no aggregations were seen in the insoluble extracts for both

proteins due to the presence of GST•Tag and S•Tag which their main functions were to

prevent the aggregation and increase the solubility of Fbp and GlpX proteins.

Based on calculation, the total expected molecular weight of the fusion proteins which

consist of GST•Tag, S•Tag and two His•Tags together with the extra bases between the

tagged proteins was 37 kDa. The native GlpX molecular weight is around 34.81 kDa,

hence, the expected size of the tagged GlpX is 71.81 kDa. After purification, the tagged

GlpX was about 73 kDa which very close to the expected size. However, the native

Fbp molecular weight is around 77.23 kDa, hence, the total expected size of tagged Fbp

is 114.23 kDa. After purification, the tagged Fbp was 115 kDa seen in SDS-PAGE and

this also was very close to the estimated size.


150

4.3.3 Assay of the purified Fbp and GlpX recombinant fusion proteins

Using the LIC cloning system, a specific cleavage site is engineered between the N-

terminal tags and the fused proteins. Hence, all N-terminal tags proteins (GST•Tag,

His•Tag, and S•Tag) should be removed by treatment with enterokinase. Several

attempts to remove the tags from the dialyzed Fbp-fusion and GlpX-fusion proteins

were carried out with various optimizations and conditions, but with no success.

Although the reason is not clear, it might be due to remaining imidazole even after

extensive dialysis.

Both JB 108 strains which harbour the plasmids that have the fbp or glpX genes, were

plated onto M9 glycerol (+Tet) plates, one with IPTG and the other one without IPTG.

As expected no growth occurred on the glycerol plate without IPTG due to the fbp or

glpX genes were under the control of the T7 promoter which only allows for expression

after induction with IPTG. However, the cells were able to thrive on the glycerol plate

supplemented with IPTG, indicating that the fbp or glpX genes were able to complement

the fbp mutation even though both the Fbp and GlpX enzymes were expressed as

recombinant fusion proteins. This provided confirmation that the tags on the fusion

proteins do not interfere with the FBPase activity of the Fbp and GlpX enzymes.

Also, both purified Fbp and GlpX were assayed the same way as described before in

section 2.7.6, however no activity were detected for Fbp or GlpX. A similar problem

was also reported by Movahedzadeh et al, (2004) who found that purification of the

recombinant M. tuberculosis GlpX led to loss of FBPase activity. This was due to

nitrilotriacetic acid (NTA) may have the ability to absorb metals and therefore

purification of a protein that is dependent on a metal for activity can result in loss of the

activity (Terpe, 2003). This problem was solved by transforming the expression vectors

that contained the fbp or glpX genes into the strain JB108, allowing preparation of crude

extracts, and assay of the enzymes as described previously in section 2.7.6. Both

recombinant tagged Fbp and GlpX proteins had FBPase activity indicating that the

fusion tags did not inhibit FBPase activity. However, there were small differences in

the activity of the native and tagged GlpX toword the respond to the effectors and this

was explained in section 4.2.2.14.

http://mic.sgmjournals.org/search?author1=F.+Movahedzadeh&sortspec=date&submit=Submit


151

4.4 Conclusion

The main goal of using Gateway expression system was to express and purify GlpX

class II and Fbp class III. The BP and LR reactions appeared to work successfully as

mentioned before. However, the sequence results were disappointments which resulted

in a decision to stop using this expression system and alternative cloning strategies were

being investigated at the same time and attention turned to one of them. Although, this

problem was reported by another student in the laboratory who was cloning different

genes at the same time. This problem was solved using LIC cloning system and both

proteins were expressed and purified, but the purified GlpX and Fbp enzymes were not

active. It is presumed that purification system Bug Buster®

Ni-NTA His•Bind is not

suitable for enzymes that depend on metal for their activity. Genetic complementation

results of both recombinant GlpX and Fbp enzymes confirm this hypothesis and both

enzymes were able to complement the JB108 mutant. Also, it was shown that Fbp

protein encodes for FBPase and this is first study reported Fbp class III enzyme in

clostridia. Moreover, the enzymes assay results were consistent with genetic

complementation that both proteins indeed encode for FBPase activity, despite the large

tag protein attached in the N-terminal. The native GlpX and recombinant fusion GlpX

assay results were almost alike. However, it seems that the large tag protein does affect

the metal bind site. Further analysis needed to be carried out in order to determine the

physiological function of glpX using RT-PCR and proteomics analysis.

Transcriptional analysis

152

Chapter 5:

5 Transcriptional analysis


153

5.1 Reverse transcriptase- Polymerase chain reaction (RT-PCR)

5.1.1 Introduction

The RT-PCR reaction consists of two amplification steps. The first one is reverse

transcription of RNA which can be performed by a primer that anneals to the RNA

template to create a complementary DNA (cDNA) by using the reverse transcriptase

(RT) enzyme, hence, this reaction is termed the RT reaction. The second step is PCR

amplification which can be performed by using any DNA polymerase enzyme (Newton

and Graham, 1994).

5.1.2 Results

5.1.2.1 Determination the expression of glpX and hprK

The unique gene arrangement that has been proposed in C. acetobutylicum by Tangney

et al, (2003) in which glpX is linked to hprK, is not found in other low GC Gram-

positive bacteria. In all the other bacteria, the identified hprK genes are linked to an lgt

gene that codes for a prolipoprotein diacylglyceryl transferase enzyme (Tangney et al.,

2003; Boel et al., 2003). Also the hprK gene overlaps with a gene that encodes for 5-

formyltetrahydrofolate cyclo-ligase, an enzyme that forms ADP, phosphate, and 5,10

methenyltetrahydrofolate via the hydrolysis of ATP and 5-formyltetrahydrofolate as per

the reaction equation (Greenberg et al., 1965).

ATP + 5-formyltetrahydrofolate ADP + phosphate + 5,10 methenyltetrahydrofolate.

In C. acetobutylicum FBP is needed for the activity of HPr kinase/phosphorylase which

phosphorylates HPr at the Ser-46 site and GlpX hydrolyses FBP. Therefore, GlpX

might be involved in regulating the kinase activity of HPrK/P which in turn would

regulate carbon catabolite repression (Tangney et al., 2003). It is very important to

prove this unique gene arrangement by experimental evidence. Therefore, an RT-PCR

was carried out using RNAglpX primers (Table ‎2.5 and Table ‎5.1) which amplify a

fragment of 369 bp between the 3` end of glpX and the 5` end of hprK genes. Also,

another RT-PCR was carried out using RNAhprK primers (Table ‎2.5 and Table ‎5.2)

which amplify the overlap fragment of 316 bp between hprK and the gene that encodes

for 5-formyltetrahydrofolate cyclo-ligase (Figure ‎5.1).


154

Table ‎5.1: Locations of RNAglpX primers on the sequences of cac1088-cac1089

(glpX-hprK genes).

Bases underlined represent the locations of RNAglpX primers. Bases highlighted in

green and red represent the start and stop codon respectively. Obtained from National

Center for Biotechnology Information.

http://www.ncbi.nlm.nih.gov//nuccore/NC_003030.1?report=fasta&from=1257296&to

=1258210

glpX start codon

ATGTTTGATAATGATATATCCATGAGTTTAGTAAGAGTAACAGAAGCAGCAGCACTACAATCTTCAAAGT

ATATGGGAAGAGGAGATAAAATTGGAGCTGATCAAGCAGCAGTAGATGGAATGGAGAAGGCATTTAGTTT

TATGCCAGTAAGAGGCCAGGTTGTAATAGGAGAGGGAGAACTTGATGAAGCTCCTATGCTTTATATAGGT

CAAAAGCTTGGTATGGGAAAAGACTATATGCCTGAAATGGATATAGCAGTAGATCCTTTAGATGGAACGA

TTTTAATTTCTAAGGGACTACCTAATGCAATAGCAGTAATAGCAATGGGACCAAAAGGAAGTTTACTTCA

TGCCCCAGATATGTATATGAAGAAAATTGTTGTGGGACCTGGAGCAAAAGGTGCTATAGATATAAATAAA

TCTCCTGAAGAGAATATTTTAAATGTAGCAAAGGCATTAAACAAGGACATATCTGAATTAACAGTTATAG

TTCAAGAAAGAGAAAGACATGACTACATAGTAAAAGCAGCTATAGAAGTTGGAGCAAGAGTTAAGCTATT

TGGTGAGGGCGATGTTGCAGCTGCACTTGCTTGTGGTTTTGAAGATACTGGAATAGACATACTTATGGGA

ATTGGAGGAGCTCCAGAAGGAGTTATAGCCGCAGCAGCTATCAAGTGCATGGGCGGAGAAATGCAGGCTC

AGCTTATACCTCATACTCAGGAAGAAATAGATAGATGTCACAAAATGGGAATAGATGATGTAAATAAAAT

TTTCATGATAGATGATTTAGTTAAAAGTGATAATGTGTTTTTTGCAGCTACAGCAATAACAGAATGTGAT

CTTCTTAAGGGCATAGTATTTTCTAAAAATGAACGTGCAAAAACCCATTCCATATTAATGAGATCTAAAA

glpX stop codon

CTGGTACAATAAGATTTGTTGAAGCTATTCATGACTTGAATAAAAGTAAATTAGTGGTAGAATAAAATTA

hprK start codon

AAATGTTCGATTTACGGCGAAGGGGGATAGTAAATGCAGGTAAGTATTGAAGATATAATAGAAAATCTCG

ATTTAGAGGTCTTGGTTAAGGGGAAAGATGGAATAAAACTGGGGTTAAGTGATATAAA

CAGACCAGGACTACAGTTTG

RNAglpX forward primer

and GATEfbp reverse primer

RNAglpX reverse primer


http://www.ncbi.nlm.nih.gov/nuccore/NC_003030.1?report=fasta&from=1257296&to=1258210



155

Table ‎5.2: Locations of RNAhprK primers on the sequences of cac1089-cac1090

(hprK-5-formyltetrahydrofolate cyclo-ligase genes).

Bases underlined represent the locations of RNAhprK primers. Bases highlighted in

green and red represent the start and stop codon. The overlap region is in bold.

Obtained from National Center for Biotechnology Information.

http://www.ncbi.nlm.nih.gov//nuccore/NC_003030.1?report=fasta&from=1257296&to

=1258210

hprK strat codon

ATGCAGGTAAGTATTGAAGATATAATAGAAAATCTCGATTTAGAGGTCTTGGTTAAGGGGAAAGATGGAA

TAAAACTGGGGTTAAGTGATATAAACAGACCAGGACTACAGTTTGCCGGATTCTATGATTATTTTGGCAA

TGAAAGAGTGCAGGTTATAGGAAAGGCTGAGTGGAGTTTTCTAAATGCAATGCCTCCAGAAATAAGAGAA

AAAAGAATTAGAAAATATTTTCAATTTGAAACACCATGTATTGTTCTTGCAAGAGGATTGAAACCACAAA

AGGAACTTCTTGATTGTTCCAAAGAGTACAATAGGTGGCTTTTAAGGTCAAAGGCTCAAACTACTAGATT

TATAAATAAAATAATGAACTATCTTGATGACAAGCTGGCTCCTGAAACAAGAATTCATGGAGTGTTAGTT

GATATCTATGGATTAGGTATACTAATCACTGGAGAAAGTGGAATCGGAAAAAGTGAAACTGCACTTGAGC

TCATAAAAAGAGGGCATAGATTGGTTGCAGATGATGCTGTAGATATAAAAGAAATTGAATCAGTTCTTGT

TGGAAAATCACCATACATAACTTCTGGTATGCTTGAGGTTAGAGGAATGGGAATAATAGATGTTCCAGCA

CTTTATGGTCTTAGTTCTGTTTTGTCAGAAAAGAATATAAATCTTGTAATATACCTTGAACAATGGAAAG

AGGGAAGAGACTACGATAGACTTGGTACAGAT

GATGAACACATAAAAATTTTAAATATTCCTGTGAGAAAAATGACGCTGCCTATACGTCCAGGGAGGAATG

TTGCTGTTATAATAGAGGCAGCAGCTGCTAATTATAGATATAATTTAAGCAGTAAAATATCTCCTGTAGA

TACTATTAATAAGAGAATTGAAGAGTCTACAAATTATGATTAATAGTAAAAAAGAACTTAGGAAAAATA

TGGTTTTAAAAAGGGATTCTTTAGAGCCAGATTTAAAATCAATAAAGGATAATATGATTTATAATAAAGT

CATAAATAGCATACAATATAAAAGTGCTAATAATATATTTGTGTTCGTTAGTTACAAAAGTGAAGTTGAT

ACTCATAATATAATAAG

AACAGCAATTGCAGATGGAAAAAAAGTTTTTGTGCCGAAGGTTATTTCCAAGG

RNAhprk forward primer


RNAhprk reverse primer





156

Figure ‎5.1: Illustration of glpX, hprK and 5-formyltetrahydrofolate cyclo-ligase

gene arrangement.

The blue box is an open reading frame located between glpX and hprK genes that would

be amplified by RNAglpX primers, the red box is an overlap region located between

hprK and 5-formyltetrahydrofolate cyclo-ligase genes that would be amplified by

RNAhprK primers.

First, normal PCR using a DNA template was carried out using the reverse and forward

RNAglpX primers and reverse and forward RNAhprK primers to check their ability to

amplify the right fragments and the best annealing temperatures (Figure ‎5.2). Three

different annealing temperatures were tested for each gene; for the RNAglpX primers

51, 53 and 55°C were tested, only 53 and 55°C were able to anneal the primers and

amplify the fragment between the the 3` end of glpX and the 5` end of hprK genes to

give a product of 369 bp (Figure 5.2). However, for the RNAhprK primers 53, 55 and

57°C were tested, and only 53 and 57°C were able to anneal and amplify the overlap

fragment between hprK and the 5-formyltetrahydrofolate cyclo-ligase gene to give a

product of 316 bp (Figure 5.2). The agarose gel showed that both annealing

temperatures for each fragment can be used in the cDNA amplification step of the RT-

PCR (Figure 5.2). Also, the primer dimers issue needed to be solved to prevent

interference with the results due to them being are very close in size to the PCR product

(Figure 5.2).


157

Figure ‎5.2: Agarose gel electrophoresis of PCR amplification of fragments between

hprK, glpX and a gene that encodes for 5-formyltetrahydrofolate cyclo-ligase.

The RNAglpX primers amplify a fragment between the 3` end of glpX and the 5` end

of hprK genes and the size of the PCR product was around 369 bp (lane 2 for 53°C and

lane 3 for 55°C). The RNAhprK primers which amplify the overlap fragment between

hprK and a gene that encodes for 5-formyltetrahydrofolate cyclo-ligase and the PCR

product was around 316 bp (lane 4 for 55°C and lane 5 for 57°C). The lane 1 represents

Hyperladder I (Bioline).

1 2 3 4 5

0.4kbp_

10kbp_

0.2kbp_


158

5.1.2.2 QIAGEN® One-Step RT-PCR Kit

The RNA template was purified as described in materials and methods section ‎2.10.1,

from C. acetobutylicum grown in glucose minimal medium (Figure ‎5.3). Several

attempts were made to amplify these fragments using the QIAGEN® One-Step RT-PCR

Kit, such as different amount of RNA template (1, 2, and 4 µl) (Figure 5.4), adding

RNase inhibitor into the reaction mix to protect from degradation and extend the life

time of the RNA template. Also the same different annealing temperatures that were

used before to amplify each fragment were tested for the cDNA amplification step of

the RT-PCR. Despite these various optimisations, no products were obtained from the

RT-PCR reaction and only primer dimer was observed (Figure ‎5.4). Moreover, the

QIAGEN® One-Step RT-PCR Kit does not come with a control reaction to test the

efficiency of the kit.

Figure ‎5.3: Agarose gel of purified total RNA from a culture of C. acetobutylicum

grown in glucose minimal medium.

Lane 1 and 2 represent duplicate sample of the total purified RNA.

1 2

16S rRNA-

23S rRNA-


159

Figure ‎5.4: Agarose gel electrophoresis of RT-PCR attempt to amplify fragments

between hprK, glpX and a gene that encodes for 5-formyltetrahydrofolate cyclo-

ligase.

Attempt to amplify fragment between the downstream of glpX and the upstream of hprK

genes using these different amounts of RNA template, 1µl for the lane 2, 2µl for the

lane 3, and 4µl for the lane 4. Lanes 5 to 7, represent the attempt to amplify the overlap

fragment between hprK and a gene that encodes for 5-formyltetrahydrofolate cyclo-

ligase using the same different amounts of RNA template. The lane 1 represents the

Hyperladder (Bioline).

1 2 3 4 5 6 7

0.4kbp_

10kbp_

0.2kbp_


160

5.1.2.3 One Step RT-PCR Master Mix Kit from Novagen®

Therefore, another kit, the One Step RT-PCR Master Mix Kit from Novagen® was used

to generate cDNA and then PCR product as described in the materials and methods

section ‎2.10.3. This kit comes with a control reaction to test the efficiency of the kit

and also it can detect very small amounts of RNA, less than 2 ng, which makes it more

sensitive. First, a control reaction to test the kit was carried out using the RT-PCR

control primers and a positive RNA control transcribed product from the human gene

G3PDH (glyceraldehyde 3-phosphate dehydrogenase) which is a housekeeping gene in

human tissue (Barber et al., 2005). The reaction mix was set up as described in

materials and method section ‎2.10.3. A positive RNA control (2 µl) and 16.5 µl RNAs-

free water to reach the reaction total volume of 50 µl. As expected a RT-PCR product

of 450 bp was detected in the agarose gel (Figure ‎5.5).

To prove the unique gene arrangement, two RT-PCR reactions were then carried out

using RNAglpX primers which amplify a fragment between 3` end of glpX and the 5`

end of hprK genes, and RNAhprK primers which amplify the overlap fragment between

hprK and the 5-formyltetrahydrofolate cyclo-ligase gene. The reaction mix was

prepared as described in the materials and methods section ‎2.10.3, however, to

overcome the primer dimer issue, primer concentrations were diluted to 10 pmol/µl (10

-fold) and the amount of RNA template used was 9 µl. A PCR product of size around

300 bp was detected in the agarose gel for each of the reactions, which indicates that

glpX and hprK are expressed on one polycistronic m-RNA and also the same results

were seen for hprK and the 5-formyltetrahydrofolate cyclo-ligase gene. Therefore, all

these glpX, hprK and 5-formyltetrahydrofolate cyclo-ligase genes are expressed as an

operon. Also, a control reaction was carried out with normal PCR reaction (no reverse

transcriptase step) using the same primers (RNAglpX) to detect any trace of DNA in the

purified RNA. However, no amplification was detected indicating that no DNA was

present in the purified RNA (Figure ‎5.6).


161

Figure ‎5.5: Agarose gel electrophoresis of a control RT-PCR reaction from the

Novagen® One Step RT-PCR Master Mix Kit.

The lanes 2, 3 represent two control RT-PCR products of size 450 bp. Lane 1,

represents the Hyperladder I (Bioline).


162

Figure ‎5.6: Agarose gel electrophoresis of RT-PCR reaction to amplify fragments

between hprK, glpX and a gene that encodes for 5-formyltetrahydrofolate cyclo-

ligase.

The lane 2, and 3 represent amplified fragments between the 3` end of glpX and the 5`

end of hprK genes (300 bp). Lanes 4 and 5 represent the amplified the overlap fragment

between hprK and a putative gene that encodes for 5-formyltetrahydrofolate cyclo-

ligase and the control reaction mix (normal PCR), respectively. The lane 1 represents

the Hyperladder I (Bioline).


163

5.2 Discussion

The kinase activity of the bifunctional HPr kinase/phosphorylase is dependent on FBP.

HPrK/P is phospholyrates the HPr at serine-46 site in an ATP dependant manner to

participate in carbon catabolite repression. However, the putative hprK gene in C.

acetobutylicum is located downstream of a gene that encode for GlpX which is a class II

FBPase enzyme (Tangney et al., 2003). This gene arrangement is exclusive to C.

acetobutylicum which might indicate the unique role of GlpX in regulating the activity

of HPr kinase, and this in turn would effect carbon catabolite repression. To confirm

how the expression of the genes is organised, RT-PCR analysis was carried out using

specific primers as mentioned previously. The RNA was extracted and purified from C.

acetobutylicum grown on glucose minimal medium conditions in which it would be

expected that HPrK would be expressed. Several attempts to amplify these fragments

using QIAGEN® One-Step RT-PCR, a kit that can detect amounts of RNA less than 50

ng, however no PCR products were seen on the gel. Therefore, RT-PCR analysis was

done using another kit, the One Step RT-PCR Master Mix Kit from Novagen® which

can detect very small amounts of RNA, less than 2 ng. The results showed that both

regions were amplified and detected on agarose gel, indicate that GlpX, HPr kinase and

the gene that encodes for 5-formyltetrahydrofolate cyclo-ligase were expressed under

glucose condition.

In B. subtilis the hprK was reported to be weak constitutive expressed gene that does

not depend on the growth condition (Hanson et al., 2002). Therefore, Northing blotting

cannot be used due to the fact that this technique is not sensitive enough to detect a

weak gene expression (Rapley and Manning, 1998). This might explain why m-RNA of

hprK-glpX cannot be detected by standard RT-PCR kit such as QIAGEN® One-Step

RT-PCR and was only detected by very sensitive RT-PCR kit which was One Step RT-

PCR Master Mix Kit from Novagen®

. Moreover, these results shown, glpX, hprK and

the 5-formyltetrahydrofolate cyclo-ligase gene were expressed together in one

polycistronic m-RNA. In prokaryotes genes that are functionally related are usually

expressed as a polycistronic m-RNA as seen for the E. coli lac operon in which all the

three genes that responsible for uptake and utilization of lactose are expressed as one

polycistronic m-RNA (Vilar et al., 2003).


164

Therefore, the RT-PCR results imply that the unique gene arrangement of the C.

acetobutylicum glpX, hprK and 5-formyltetrahydrofolate cyclo-ligase genes might

indicate that they have a related function in regulating carbon catabolite repression in

this industrial important strain. The physiological function of 5-formyltetrahydrofolate

cyclo-ligase is still unknown in prokaryotes. However, a recent study indicates that this

protein is necessary in thiamine (Vitamin B1) metabolism (Pribat et al., 2011). Also,

the expression of glpX in cells grown in glucose minimal medium indicates that this

gene is not simply an enzyme of the gluconeogensis pathway which enables cells to

grow on gluconeogenic substrate such as glycerol as reported by others (Rittmann et al.,

2003; Movahedzadeh et al., 2004; Jules et al., 2009). Instead, glpX in C.

acetobutylicum can be suggested to have a specific role in regulating the activity of HPr

kinase. Also, proteomic analysis was carried out in order to confirm whether glpX is

expressed or not when the cells are grown on glucose as a sole carbon source. The

results showed that liquid chromatography electrospray ionization tandem mass

spectrometry was able to detect 439 proteins and GlpX was among the proteins which

indicate that glpX gene is expressed during growth on a glycolyte substrate such as

glucose which is consistent with the RT-PCR result. However no Fbp was detected

under these conditions and this indicates that Fbp might have role gluconeogenic

pathway.


165

5.3 Conclusion

C. acetobutylicum total RNA was purified from a culture grown on glucose minimal

medium. Then two RT-PCR reactions were carried out using RNAglpX and RNAfbp

primers to amplified regions between the 3` end of glpX and the 5` end of hprK genes

and the overlap region between hprK and the 5-formyltetrahydrofolate cyclo-ligase

gene. The RT-PCR results indicate that GlpX is expressed under glucose growth

conditions and expressed as a polycistronic m-RNA together with hprK and the 5-

formyltetrahydrofolate cyclo-ligase gene which also imply that these genes are

expressed as an operon. Furthermore, proteomic analysis was compatible with RT-PCR

results. Based on these results, GlpX seems to be expressed constitutively due to it

linked to hprK which is constitutive expressed gene that does not depend on the growth

condition and needed for CCR. Therefore, GlpX might play a vital role in CCR.

General discussion

166

Chapter 6:

6 General discussion

General discussion

167

6.1 The importance of intracellular FBP levels in CCR and strain improvements

In this project the glpX and fbp genes of C. acetobutylicum were shown by genetic

complementation and enzyme assay to encode for FBPase enzymes. Both GlpX and

Fbp protiens were inhibited by 1 mM inorganic phosphate and the recombinant tagged

Fbp had 25 fold higher activity than the recombinant tagged GlpX. To investigate this

further, proteomic and RT-PCR analyses were carried out. In proteomic analysis, the

Fbp protein was not found under glucose growth conditions, but, GlpX was detected.

RT-PCR results showned that glpX does indeed express on one polycistronic m-RNA

together with hprK and 5-formyltetrahydrofolate cyclo-ligase genes under glucose

growth conditions. These results are consistent with the GlpX would appear to have a

different role, whereas, the Fbp enzyme appear to involve in utilizing gluconeogenic

substrates, Given the position of the glpX gene in C. acetobutylicum genome, proteomic

and RT-PCR analyses which work towards a role in regulating intracellular FBP

concentration.

As mentioned before, FBP is a key metabolite effector that can enhance binding of the

CcpA-HPr-P-Ser complex to a cre site which in turn results in repression or activation

of an operon or gene (Henkin, 1996; Presecan-Siedel et al., 1999; Schumacher et al.,

2007; Antunes et al., 2010). In B. subtilis, disruption of the gene encoding

phosphofructokinase, which prevented the production of FBP, resulted in no CCR and

the growth was impaired (Nihashi and Fujita, 1984). These results indicated how FBP

is important in mediating the level of CCR in Gram positive bacteria. Intracellular FBP

concentration has vital role in regulating the activity of HPr kinase and in turn CCR in

low GC Gram positive bacteria such as B. subtilis (Singh et al., 2008; Deutscher et al.,

2006). In this microorganism the highest FBP concentration was detected in vivo when

cells were grown on glucose which is the preferable sugar that exerts the strongest CCR

effect (Singh et al., 2008; Nihashi and Fujita, 1984).

Singh et al, (2008) have tested the effects of many different substrates on CCR. These

tested substrates such as ribose, sucrose, glucose, and fructose have the ability to exert

CCR and the repression caused by these substrates was quantified using the activity of

β-xylosidase (XynB) which is involved in xylose utilization. In the absence of xylose, a

very low β-xylosidase activity was detectable (14 U/mg), however, in the presence of

xylose, a high β-xylosidase was detectable (945 U/mg) demonstrating that the synthesis

of the enzyme was induced.

General discussion

168

In the quantitative analysis, cells were grown on xylose together with a repressing sugar

to find out how XynB synthesis was affected, and intracellular FBP concentration was

also measured. Glucose exerted the strongest repression on XynB activity which was

reduced to about 7 U/mg. However, maltose, sucrose and ribose repressed the activity

to about 437, 126 and 497 U/mg respectively which was very weak repression

compared to glucose. These results, however, indicated that not only glucose, but also

other substrates, can exert CCR (Singh et al., 2008). Moreover, the amount of

intracellular FBP during growth in the presence of glucose, maltose, sucrose and ribose

were around 14.1, 10.7, 11.5, 6.5 mM respectively (Singh et al., 2008). By comparison,

under gluconeogenic growth conditions such as on malate intracellular FBP in B.

subtilis was found to be very low (around 1.5 mM) (Kleijn et al., 2009). The

implication of the results is that a higher FBP concentration increases the activity of

HPr kinase which phosphorylates more HPr protein at the serine 46 site to cause CCR,

while substrates which generate a low level of intracellular FBP do not exert CCR

(Carlos et al, 2003; Singh et al., 2008).

For developing a successful industrial strain, clarification is needed on metabolic and

regulatory networks of this strain (Dobson et al., 2011). Although several attempts to

control metabolic pathway in C. acetobutylicum in order to increase solvent production

were carried out. Recently, the ack gene which encodes for acetate kinase that involve

in acetate formation was disrupted using the ClosTron system with the aim to increase

the butanol production by reducing acetate and acetone formation (Kuit et al., 2012).

Although, acetate production in the mutant strain was reduced by more than 80%

compare to the wild type, however, the acetone production was not effected in the

mutant strain. This indicates that another acetate producing pathway might be operated

in C. acetobutylicum (Kuit et al., 2012).

Therefore intracellular FBP concentration might be used as an important global

regulatory signal to control CCR in Gram positive bacteria such as C. acetobutylicum,

in which a similar regulatory mechanism appears to be present. It has been reported that

C. acetobutylicum and C. butyricum AKR102a are able to grow on crude glycerol

derived from biodiesel (Carlos et al., 2003; Ringel et al., 2011).

General discussion

169

As mentioned before, cloning and overexpression of either the fbp or glpX gene of C.

acetobutylicum allowed an E. coli fbp mutant to thrive in glycerol minimal media.

Therefore, overexpression of either Fbp or GlpX in C. acetobutylicum might lead to an

increase in growth rate and the utilization efficiency of crude glycerol and thus increase

butanol production. At the same time, overexpression of either gene might also result in

a drop in the intracellular FBP concentration which would most likely relieve CCR

when the cells grow on multiple carbon sources and increase the ability of the cells to

utilize multiple carbon sources. Therefore, this might solve the problem of CCR

excreted via using fermentation substrates that contain mixture of several carbon

sources concurrently.

Also, this metabolic engineered strain probably can be used in the current biodiesel

industry not only to increase the profit but solve the excessive crude glycerol formed

from the production of biodiesel. Although genomic DNA of the recently isolated C.

butyricum AKR102a strain has not been sequenced yet, this strain might contain genes

encoding fbp in order to grow on glycerol. Therefore, the same proposed metabolic

engineering techniques mentioned above for C. acetobutylicum might be used to

increase the utilization efficiency of crude glycerol and production of 1,3-PD. The

availability of genomic DNA sequence may assist in confirming these hypotheses by

allowing characterization of the genes that encode for FBPase activity in this strain and

identifying the locations of those genes.

It has been reported that knockout of the hprK gene in Gram positive bacteria such as

Streptococcus mutans and B. subtilis led to impaired growth even though the CCR was

relieved (Deutscher et al., 1994; Zeng et al., 2010). Also a knockout of ccpA in C.

acetobutylicum apparently had the same result (Ren et al., 2010). These effects are

presumably due to several genes being positively regulated by these CCR elements and

any mutations introduced to these elements may lead to a defect in the growth. An

alternative way to relieve CCR in Gram positive bacteria without disturbing growth rate

may be to reduce the intracellular FBP concentration by overexpressing a gene that

encodes for FBPase to mimic the conditions when the bacteria are grown on unpreferred

substrates such as glycerol. Knockout of a gene that encodes FBPase might result in

several phenotypes, depending on how many genes encode for FBPase enzyme in the

bacterium.

General discussion

170

For example, in a bacterium that has only one gene that encodes for FBPase is most

likely to involve in gluconeogenesis pathway and the knockout of this gene would be

expected to result in the mutant strain being unable to grow on gluconeogenic substrate

such as glycerol. This phenomenon was reported in C. glutamicum and M. tuberculosis

in which there is only one gene that encodes for FBPase enzyme (Movahedzadeh et al.,

2004; Rittmann et al., 2003). However, if the bacteria contain two genes or more

encoding for FBPase enzyme, one of these genes may encode for the major FBPase

involved in the gluconeogenesis pathway and the others might have different

physiological functions. In E. coli for example, knockout of the major fbp gene which

encodes for the class I FBPase enzyme created the ∆fbp strain which is unable to grow

on gluconeogenic substrates such as glycerol, whereas the glpX mutant do not show any

change in the phenotype (Donahue et al., 2000). Therefore, it is most likely that

knockout of the fbp gene in C. acetobutylicum would result in a strain that is unable to

grow on a gluconeogenic substrate, while, disrupting of the glpX gene might generate a

strain with very tight CCR due to high level of intracellular FBP concentration.

General discussion

171

6.2 Conclusions and future work

This study has demonstrated that C. acetobutylicum contains two genes encoding for

class II and III FBPase enzymes. In order to confirm their physiological functions,

three mutants need to be constructed, ∆fbp, ∆glpX, and a double mutant (∆fbp and

∆glpX) and tested with different carbon sources to detect any variations in the growth

phenotype. Also the intracellular FBP concentration of these mutants together with the

wild type strain need to be tested to determine whether this is affected by growth with

various carbon sources or not. These experiments would shed some light on how GlpX

can interfere with CCR and also confirm the proposed function of Fbp. These findings

could form the basis of constructing strains that would show optimum performance in

fermentations using a range of substrates and conditions.

Appendix

172

7 Appendix

Appendix

173

7.1 Sequence of the recombinant fusion tags

Obtained from Novagen (2010) LIC

Cloning User Manual.

His-tag sequence: (molecular weight = 900 Da) HHHHHH.

S-tag sequence: (molecular weight = 4500 Da) LGTAAALPGAGHMAS.

GTS-tag sequence: (molecular weight = 26 KDa)

MKLFYKPGACSLASHITLRESGKDFTLVSVDLMKKRLENGDDYFAVNPKGQVP

ALLLDDGTLLTEGVAIMQYLADSVPDRQLLAPVNSISRYKTIEWLNYIATELHK

GFTPLFRPDTPEEYKSTVRAQLEKKLQYVNEALKDEHWICGQRFTIADAYLFTV

LRWAYAVKLNLEGLEHIAAFMQRMAERPEVQDALSAEGLK

Appendix

174

7.2 Bioline Hyperladders

DNA molecular weight markers obtained from:

http://www.bioline.com/h_ladderguide.asp

http://www.bioline.com/h_ladderguide.asp

Appendix

175

7.3 HyperPAGE Prestained Protein Marker

Prestained protein markers obtained from Bioline:

http://www.bioline.com/documents/product_inserts/HyperPAGE.pdf#zoom=130

http://www.bioline.com/documents/product_inserts/HyperPAGE.pdf#zoom=130

Appendix

176

7.4 pCR 2.1 TOPO Cloning Vector

Obtained from Invitrogen (2006) TOPO TA


http://www.invitrogen.com/content/sfs/manuals/topota_man.pdf

http://www.invitrogen.com/content/sfs/manuals/topota_man.pdf

Appendix

177

7.5 The‎donor‎vector‎pDONR™221.

Obtained from Invitrogen (2009) cloning user manual.

http://tools.invitrogen.com/content/sfs/manuals/gateway_pdonr_vectors.pdf

http://tools.invitrogen.com/content/sfs/manuals/gateway_pdonr_vectors.pdf

Appendix

178

7.6 The Gateway® Nova pET-60-DEST™‎vector

Obtained from Novagen (2009) Gateway


http://www.merck-chemicals.com/united-kingdom/life-science-research/gateway-nova-

pet60destexpressionsystem/EMD_BIO71863/p_C3Ob.s1OvmAAAAEjEhx9.zLX;sid=l

qvX0FI_JYLd0B0mb5O6h_r_ESjFINyxuku9d2oR9QHR7kIjlXIq6Hs4t1_OlJ7vpTHHz

FHmESjFINW8hFvHzFHm?attachments=USP

http://www.merck-chemicals.com/united-kingdom/life-science-research/gateway-nova-pet60destexpressionsystem/EMD_BIO71863/p_C3Ob.s1OvmAAAAEjEhx9.zLX;sid=lqvX0FI_JYLd0B0mb5O6h_r_ESjFINyxuku9d2oR9QHR7kIjlXIq6Hs4t1_OlJ7vpTHHzFHmESjFINW8hFvHzFHm?attachments=USP




Appendix

179

7.7 The pET-41 Ek/LIC vector

Obtained from Novagen (2010) LIC


http://www.merck-chemicals.com/united-kingdom/life-science-research/pet-41-ek-lic-

vector-kit/EMD_BIO-71071/p_BICb.s1O130AAAEj5Rt9.zLX?attachments=USP

http://www.merck-chemicals.com/united-kingdom/life-science-research/pet-41-ek-lic-vector-kit/EMD_BIO-71071/p_BICb.s1O130AAAEj5Rt9.zLX?attachments=USP

http://www.merck-chemicals.com/united-kingdom/life-science-research/pet-41-ek-lic-vector-kit/EMD_BIO-71071/p_BICb.s1O130AAAEj5Rt9.zLX?attachments=USP

Appendix

180

7.8 Amino acids abbreviation

Obtained from the LabRat.com (2005).

http://www.thelabrat.com/protocols/aminoacidtable.shtml

Name Abbreviation

3-Letter 1-Letter

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C

Glutamic Acid Glu E

Glutamine Gln Q

Glycine Gly G

Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

http://www.thelabrat.com/protocols/aminoacidtable.shtml

Appendix

181

7.9 Calibration curve of protein

Typical calibration curve for estimation of concentration of protein.

Appendix

182

7.10 FBPase enzyme assay curve

Typical FBPase enzyme assay curve for calculating the FBPase specific activity.

Appendix

183

7.11 Proteomic analysis of FBPase class II (GlpX) protein

Typical output from liquid chromatography electrospray ionization tandem mass

spectrometry (LC/ESI-MS/MS).

Match to: gi|15894373 (glpX) Score: 92

fructose 1,6-bisphosphatase II [Clostridium acetobutylicum ATCC 824]

Found in search of DATA.TXT

Nominal mass (Mr): 35015; Calculated pI value: 5.08

NCBI BLAST search of gi|15894373 against nr

Unformatted sequence string for pasting into other applications

Fixed modifications: Carbamidomethyl (C)

Variable modifications: Oxidation (M)

Cleavage by Trypsin: cuts C-term side of KR unless next residue is P

Sequence Coverage: 7%

Matched peptides shown in Bold Red

1 MFDNDISMSL VRVTEAAALQ SSKYMGRGDK IGADQAAVDG MEKAFSFMPV

51 RGQVVIGEGE LDEAPMLYIG QKLGMGKDYM PEMDIAVDPL DGTILISKGL

101 PNAIAVIAMG PKGSLLHAPD MYMKKIVVGP GAKGAIDINK SPEENILNVA

151 KALNKDISEL TVIVQERERH DYIVKAAIEV GARVKLFGEG DVAAALACGF

201 EDTGIDILMG IGGAPEGVIA AAAIKCMGGE MQAQLIPHTQ EEIDRCHKMG

251 IDDVNKIFMI DDLVKSDNVF FAATAITECD LLKGIVFSKN ERAKTHSILM

301 RSKTGTIRFV EAIHDLNKSK LVVE

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&AUTO_FORMAT=Semiauto&CDD_SEARCH=on&CLIENT=web&COMPOSITION_BASED_STATISTICS=on&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=(none)&EXPECT=10&FILTER=L&FORMAT_BLOCK_ON_RESPAGE=None&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&GAPCOSTS=11+1&I_THRESH=0.001&LAYOUT=TwoWindows&MATRIX_NAME=BLOSUM62&NCBI_GI=on&PAGE=Proteins&PROGRAM=blastp&QUERY=MFDNDISMSLVRVTEAAALQSSKYMGRGDKIGADQAAVDGMEKAFSFMPVRGQVVIGEGELDEAPMLYIGQKLGMGKDYMPEMDIAVDPLDGTILISKGLPNAIAVIAMGPKGSLLHAPDMYMKKIVVGPGAKGAIDINKSPEENILNVAKALNKDISELTVIVQERERHDYIVKAAIEVGARVKLFGEGDVAAALACGFEDTGIDILMGIGGAPEGVIAAAAIKCMGGEMQAQLIPHTQEEIDRCHKMGIDDVNKIFMIDDLVKSDNVFFAATAITECDLLKGIVFSKNERAKTHSILMRSKTGTIRFVEAIHDLNKSKLVVE&SERVICE=plain&SET_DEFAULTS.x=9&SET_DEFAULTS.y=5&SHOW_OVERVIEW=on&WORD_SIZE=3&END_OF_HTTPGET=Yes

http://linux1.mri.sari.ac.uk/mascot/cgi/getseq.pl?C_Acetobutylicum+gi%7c15894373+seq

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Characterisation of class II and III fructose …/media/worktribe/output-191544/...Ali Abdullah Almohaish A thesis submitted in partial fulfilment of the requirements of Edinburgh